Therapeutic compositions for the treatment of dry eye disease

ABSTRACT

Described herein are materials and methods of treating dry eye disease in a subject.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No EY-12963awarded by the National Institute of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the field of ophthalmology.

BACKGROUND OF THE INVENTION

Dry Eye Disease (DED) is a relatively common condition characterized byinadequate tear film protection of the cornea. Dry eye symptoms havetraditionally been managed with eyelid hygiene, topical antibiotics(erythromycin or bacitracin ointments), oral tetracyclines(tetracycline, doxycycline, or minocycline), anti-inflammatory compounds(cyclosporine) and corticosteroids which are often time consuming,frustrating, and frequently ineffective or variably effectivetreatments.

Tens of millions of people (mostly women) are affected worldwide by dryeye. 10 million people in US are affected with severe dry eyes and morethan 3.2 million women and 1.6 million men above the age of 50 yearsbeing affected by dry eye in the US. DED is a potentially disablingdisease adversely impacting the vision-related quality of life. It leadsto ocular discomfort, a degradation in visual performance (readingspeed, contrast sensitivity), and a loss of productivity. Currenttherapeutic options are limited and costly. Topical cyclosporine-A(Restasis®) is the only approved treatment for DED in US (only). Despitethe high incidence of DED, there is currently no consistently effectivetreatment for this condition and it still remains a therapeuticchallenge. As such, there is a need for new therapeutic modalities totreat DED.

SUMMARY OF THE INVENTION

The present invention discloses a novel method for the treatment of dryeye disease in humans comprising administration of ananti-lymphangiogenic agent to the human subject. In particular, thepresent invention discloses a novel method for the treatment of dry eyedisease in humans. In some embodiments, the method comprises locallyadministering an anti-lymphangiogenic agent to the ocular surface of thehuman. Preferably, the amount of the anti-lymphangiogenic agent employedis effective to inhibit the binding of VEGF-C and/or VEGF-D ligand toVEGFR-3 or the stimulatory effect of VEGF-C and/or VEGF-D on VEGFR-3.

The present invention is based on novel evidence for the selectivegrowth of lymphatic vessels in DED cornea. Additionally, significantincrease in both caliber and extent of lymphatics in DED corneas isaccompanied by over expression of lymphangiogenic receptor VEGFR-3,further correlating DED with lymphangiogenesis.

An anti-lymphangiogenic agent of the invention is selected from thegroup consisting of: a nucleic acid molecule, an aptamer, an antisensemolecule, an RNAi molecule, a protein, a peptide, a cyclic peptide, anantibody or antibody fragment, a polysaccharide, and a small molecule.The anti-lymphangiogenic agent described herein can be administeredpurely as a prophylactic treatment to prevent dry eye disease insubjects at risk for developing dry eye disease, or as a therapeutictreatment to subjects afflicted with dry eye disease, for the purpose ofinhibiting lymphangiogenesis in the eye of a subject in need thereof.

In one preferred embodiment of the invention, the anti-lymphangiogenicagent is an inhibitor of VEGF-C or VEGF-D mediated signal transductionby VEGFR-2 or VEGFR-3. In a particularly preferred embodiment, theanti-lymphangiogenic agent is an inhibitor of VEGF-C or VEGF-D mediatedsignal transduction by VEGFR-3.

In one aspect of the invention, the inhibitor of VEGF-C or VEGF-Dmediated signal transduction by VEGFR-2 or VEGFR-3 is a molecule such asbut not restricted to an antibody, a small molecule or a peptide thatprevents binding of VEGF-C or VEGF-D to the receptors VEGFR-2 orVEGFR-3.

In another aspect of the invention, the inhibitor of VEGF-C or VEGF-Dmediated signal transduction is a VEGFR-2 or VEGFR-3 soluble receptor.Soluble receptors of VEGFR-2 or VEGFR-3 can be administered directly.Alternatively, increase in the secretion of VEGFR-2 or VEGFR-3 isaccomplished by inserting the VEGFR-2 or VEGFR-3 soluble receptors genesinto the genome of corneal cells. This could be epithelial cells,keratocytes, fibroblasts, endothelial cells, or bone marrow-derivedcells. Methods to introduce genes into a genome of a cell are well-knownin the art. Genes are introduced in the genome of corneal cells usingviral or non-viral vectors. Viral vectors include for exampleadenoviruses, retroviruses or lentiviruses. Non-viral vectors include,for example, liposomes such as cationic lipids, nanoparticles,lipoplexes and polyplexes (complexes of polymers with DNA).

In one embodiment of the invention, the anti-lymphangiogenic agent isadministered in combination with an anti-inflammatory agent such as, butnot limited to, a composition inhibiting the activity of an inflammatorycytokine selected from the group comprising IL-1, IL-17, TNF-α and IL-6.

Exemplary functional blockers of IL-1 are described in WO/2009/025763.Exemplary functional blockers of TNF-α include, but are not limited to,recombinant and/or soluble TNF-α receptors, monoclonal antibodies, andsmall molecule antagonists and/or inverse agonists. One or morecommercially-available TNF-α blocking agents are reformulated fortopical administration in this embodiment. Exemplary commercial TNF-αblocking agents used for reformulation include, but are not limited to,etanerept/Ernbrel, infliximab/Remicade, and adalimumab/Humira.

In one embodiment of the invention, the anti-lymphangiogenic agent isadministered in combination with an antiobiotic. Exemplary antibioticcompositions used for combination-therapy with antagonists ofIL-mediated inflammation include but are not limited to, amikacin,gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin,teicoplanin, vancomycin, azithromycin, clarithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, troleandomycin, amoxicillin,ampicillin, azlocillin, carbenicillin, clozacillin, dicloxacillin,flucozacillin, meziocillin, nafcillin, penicillin, piperacillin,ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,oflazacin, trovafloxacin, mafenide, sulfacetamide, sulfamethizole,sulfasalazine, sulfisoxazole, trimethoprim, cotrimoxazole,demeclocycline, soxycycline, minocycline, oxytetracycline, ortetracycline.

In one embodiment, the composition of the invention is locally appliedto the ocular tissue, alternatively the composition of the invention isapplied to the eyelids, the ocular surface, the meibomian glands or thelacrimal glands.

The composition can be in the form of a solid, a paste, an ointment, agel, a liquid, an aerosol, a mist, a polymer, a film, an emulsion, or asuspension.

Optionally, the composition further contains a compound selected fromthe group consisting of a physiological acceptable salt, poloxameranalogs with carbopol, carbopol/hydroxypropyl methyl cellulose (RP MC),carbopol-methyl cellulose, carboxymethylcellulose (CMC), hyaluronicacid, cyclodextrin, and petroleum.

In one embodiment, described herein is a method of inhibiting dry eyedisease in a subject at risk for developing dry eye disease comprisingidentifying a subject as being at risk for developing dry eye diseaseand administering an anti-lymphangiogenic agent to the subject. Theamount of the anti-lymphangiogenic agent administered to the subject ispreferably in an amount effective to inhibit the development of dry eyedisease in the subject.

In another embodiment, described herein is a method of selecting atherapeutic regimen for a subject in need thereof comprising screening asubject for one or more symptoms of dry eye disease and prescribing forthe subject administration of a composition comprising ananti-lymphangiogenic agent described herein. In another embodiment,described herein is a method of treating a subject affected with dry eyedisease comprising identifying a subject as having one or more symptomsof dry eye disease and administering a composition comprising ananti-lymphangiogenic agent to the subject.

In some embodiments, the methods described herein further compriseprescribing (or administering) a standard of care regimen for thetreatment of dry eye disease. In the context of methods describedherein, “standard of care” refers to a treatment that is generallyaccepted by clinicians for a certain type of patient diagnosed with atype of illness. For dry eye disease, for example, an aspect of theinvention is to improve standard of care therapy with co-therapy withanti-lymphangiogenic agents described herein that inhibitlymphangiogenesis.

Another aspect described herein is a method of treating a human subjectwith dry eye disease that has been hypo-responsive to a standard of caretreatment for dry eye disease comprising administering ananti-lymphangiogenic agent described herein. For example, such a methodcomprises administering to such a subject a therapeutically effectiveamount of a composition that comprises an anti-lymphangiogenic agentdescribed herein. Such a method optionally includes a step, prior to theadministering step, of selecting for treatment a subject with dry eyedisease that is hypo-responsive to a standard of care regimen for thetreatment of the dry eye disease. The term “hypo-responsive” embracessubjects that failed to respond adequately to a standard of caretreatment and subjects that initially responded, but for whom thestandard of care treatment has become less effective over time.

In one embodiment, methods described herein optionally compriseadministering a VEGFR-2 inhibitor product to the subject. The “VEGFR-2inhibitor product” can be any molecule that acts with specificity toreduce VEGF-C/VEGFR-2, VEGF-D/VEGFR-2 or VEGF/VEGFR-2 interactions,e.g., by blocking VEGF-C or VEGF-D binding to VEGFR-2, by blocking VEGFbinding to VEGFR-2 or by reducing expression of VEGFR-2. In oneembodiment, the VEGFR-2 inhibitor inhibits VEGF-C and VEGF-D binding toVEGFR-2. In another embodiment, the VEGFR-2 inhibitor inhibits bindingof VEGF to VEGFR-2. The VEGFR-2 inhibitor can be a polypeptidecomprising a soluble VEGFR-2 extracellular domain fragment (amino acids20-764 of SEQ ID NO: 51) that binds VEGF or VEGF-C or VEGF-D; VEGFR-2anti-sense polynucleotides or short-interfering RNA (siRNA);anti-VEGFR-2 antibodies; a VEGFR-2 inhibitor polypeptide comprising anantigen-binding fragment of an anti-VEGFR-2 antibody that inhibitsbinding between VEGFR-2 and VEGF or VEGF-C or VEGF-D; an aptamer thatinhibits binding between VEGFR-2 and VEGF; an aptamer that inhibitsbinding between VEGFR-2 and VEGF-C; an aptamer that inhibits bindingbetween VEGFR-2 and VEGF-D; or a fusion protein comprising the solubleVEGFR-2 polypeptide fragment fused to an immunoglobulin constant regionfragment (Fc). In some embodiments, a VEGFR-2 polypeptide fragment isfused to alkaline phosphatase (AP).

In another embodiment, the methods described herein optionally compriseadministering one or more anti-inflammatory agents to the subject. Inanother embodiments, the methods described herein optionally furthercomprise administering a tyrosine kinase inhibitor that inhibits VEGFR-2and/or VEGFR-3 activity.

Dry eye disease may be attributable to a number of factors, andtreatment of subjects who have developed dry eye disease due to avariety of specific factors is contemplated. In some variations, the DEDto be treated is DED caused by any condition other than an alloimmuneresponse. Alloimmune responses may result, for example, in some cornealtransplant patients. More specifically, in some variations, the DED tobe treated is an autoimmune DED or a DED associated with Sjogren'ssyndrome. In some variations, the DED is due to excessively fast tearevaporation (evaporative dry eyes) or inadequate tear production. Insome variations, the dry eye disease is attributable to one or morecauses selected from: aging, contact lens usage and medication usage. Insome variations, the dry eye disease is a complication of LASIKrefractive surgery. In other variations, the DED arises in a subject whohas not had eye surgery of any kind, e.g., treatment of subjects in whomthe DED is caused by LASIK surgery, corneal transplant surgery, or otherocular surgeries.

In some variations, the invention is directed to prophylaxis, to preventDED disease or its symptoms from developing. Prophylaxis may beappropriate, for example, in the context of subjects who have sufferedan eye trauma such as infection, injury, chemical exposure,inflammation, or other situations that have been shown to cause orpredispose individuals to DED. In some variations, the prophylaxis iscontinued until the trauma or the observable effects of the trauma haveresolved, or for a limited duration, e.g., 1, 2, 3, or 4 weeksthereafter.

In a preferred embodiment, the mammalian subject is a human subject.Practice of methods of the invention in other mammalian subjects,especially mammals that are conventionally used as models fordemonstrating therapeutic efficacy in humans (e.g., primate, porcine,canine, or rabbit animals), is also contemplated.

In addition to the foregoing, the following paragraphs are alsoconsidered aspects of the invention:

1. A method of treating dry eye disease (DED) in a human comprising:

administering a composition comprising at least one anti-lymphangiogenicagent and a pharmaceutically acceptable carrier the human, in an amounteffective to treat dry eye disease.

2. The method of claim 1, wherein the anti-lymphangiogenic agent isadministered to the eye of the human.

3. The method of paragraph 1, wherein the anti-lymphangiogenic agent isan inhibitor of VEGF-C or VEGF-D mediated signal transduction by VEGFR-2or VEGFR-3.

4. An anti-lymphangiogenic agent for use in the treatment of dry eyedisease.

5. Use of at least one anti-lymphangiogenic agent in a compositioncomprising a pharmaceutically acceptable carrier for administering theeye of a human, for the treatment of dry eye disease.

6. The method or use of paragraph 1 or 5, wherein the DED is anautoimmune DED or a DED associated with Sjogren's syndrome.

7. The method or use of any one of paragraphs 1-3 and 5, wherein the DEDis DED due to excessively fast tear evaporation (evaporative dry eyes)or inadequate tear production.

8. The method or use of any one of paragraphs 1-3 and 5, wherein the dryeye disease is attributable to one or more causes selected from: aging,contact lens usage and medication usage.

9. The method or use of any one of paragraphs 1-3 and 5, wherein the dryeye disease is a complication of LASIK refractive surgery.

10. The method or use of any one of paragraphs 1-3 and 5, wherein the atleast one anti-lymphangiogenic agent is purified or isolated.

11. The method or use of any one of paragraphs 1-3 and 5, wherein saidat least one anti-lymphangiogenic agent comprises a member selected fromthe group consisting of: a nucleic acid molecule, an aptamer, anantisense molecule, an RNAi molecule, a protein, a peptide, a cyclicpeptide, an antibody or antibody fragment, a polysaccharide, or a smallmolecule.

12. The method or use of any one of paragraphs 1-3 and 5-11, whereinsaid at least one anti-lymphangiogenic agent comprises a member selectedfrom the group consisting of a VEGFR-3 inhibitor, a VEGF-D inhibitor anda VEGF-C inhibitor.

13. The method or use of any one of paragraphs 1-3 and 5-11, wherein theat least one anti-lymphangiogenic agent comprises a member selected fromthe group consisting of a VEGF-C antibody, a VEGF-D antibody, a VEGF-R3antibody, and a polypeptide comprising a soluble VEGFR-3 fragment thatbinds VEGF-C or VEGF-D.

14. The method or use of paragraph 13, wherein the at least oneanti-lymphangiogenic agent comprises a VEGF-C antibody.

15. The method or use of paragraph 14, wherein the antibody comprises aheavy chain variable region set forth in amino acids 1-118 of SEQ ID NO:48.

16. The method or use of paragraph 14, wherein antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 48.

17. The method or use of paragraph 14, wherein the VEGF-C antibody isselected from the group consisting of antibodies 69D09, 103, MM0006-2E65and 193208.

18. The method or use of any one of paragraphs 1-3 and 5-11, wherein theat least one anti-lymphangiogenic agent comprises an antibody thatcompetitively inhibits the binding of antibody 69D09 to VEGF-C.

19. The method or use of any one of paragraphs 1-3 and 5-11, wherein theat least one anti-lymphangiogenic agent comprises a VEGF-D antibody.

20. The method or use of paragraph 19, wherein the VEGF-D antibody isselected from the group consisting of antibodies 2F8, 4A5(VD1), 4E10,5F12, 4H4, 3C10 28AT743.288.48, MM0001-7E79, RM0007-8C35, 78902, 78939and 90409.

21. The method or use of any one of paragraphs 1-3 and 5-11, wherein theat least one anti-lymphangiogenic agent comprises a soluble VEGFR-3fragment that binds VEGF-C or VEGF-D.

22. The method or use of any one of paragraphs 1-3 and 5-11, wherein theat least one anti-lymphangiogenic agent comprises a human or humanizedantibody.

23. The method or use of any one of paragraphs 1-3 and 5-11, wherein theat least one anti-lymphangiogenic agent comprises a VEGFR-2 inhibitor.

24. The method or use of any one of paragraphs 1-3 and 5-11 wherein theat least one anti-lympnahgiogenic agent comprises a tyorine kinaseinhibitor that inhibits the activity of VEGFR-3.

25. The method or use of any one of paragraphs 1-3 and 5-24, furthercomprising administering an anti-inflammatory agent to the subject.

26. The method or use of paragraph 25, further comprising administeringcyclosporine to the subject.

27. The method or use of any one of paragraphs 1-3 and 5-26, whereinsaid composition further comprises a molecule that inhibits an activityof an inflammatory cytokine selected from the group consisting of IL-1,IL-7, IL23, IL-6 and TNF-α.

28. The method or use of any one of paragraphs 1-3 and 5-27, wherein themethod further comprises administering an antibiotic to the human.

29. The use of any one of paragraphs 5-27 further including the use ofan antibiotic for the treatment of the dry eye disease.

30. The method or use of paragraph 28 or 29, wherein the antibiotic isselected from the group consisting of amikacin, gentamicin, kanamycin,neomycin, netilmicin, streptomycin, tobramycin, teicoplanin, vancomycin,azithromycin, clarithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, troleandomycin, amoxicillin, ampicillin,azlocillin, carbenicillin, clozacillin, dicloxacillin, flucozacillin,meziocillin, nafcillin, penicillin, piperacillin, ticarcillin,bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,oflazacin, trovafloxacin, mafenide, sulfacetamide, sulfamethizole,sulfasalazine, sulfisoxazole, trimethoprim, cotrimoxazole,demeclocycline, soxycycline, minocycline, oxytetracycline, ortetracycline.

31. The method or use of any one of paragraphs 1-3 and 5-27, wherein theeye comprises a tissue or gland in or around the eye selected from thegroup consisting of ocular tissue, eyelids of the subject, ocularsurface, meibomian gland and or lacrimal gland of the human.

32. The method of any one of paragraphs 1-3 and 5-27, wherein saidcomposition is administered topically to the eye.

33. The use according to any one of paragraphs 5-27, wherein thecomposition is formulated for topical administration.

34. The method or use of any one of paragraphs 1-3 and 5-33, whereinsaid composition is in the form of a solid, a paste, an ointment, a gel,a liquid, an aerosol, a mist, a polymer, a film, an emulsion, or asuspension.

35. The method or use of any one of paragraphs 1-3 and 5-34, wherein thecomposition further comprises a compound selected from the groupconsisting of physiological acceptable salt, poloxamer analogs withcarbopol, carbopol/hydroxypropyl methyl cellulose (RP MC),carbopol-methyl cellulose, carboxymethylcellulose (CMC), hyaluronicacid, cyclodextrin, and petroleum.

Additional aspects, features and variations of the invention will beapparent from the entirety of this application, including the detaileddescription, and all such features are intended as aspects of theinvention. It should be understood, however, that the detaileddescription and the specific examples are given by way of illustration,and that the many various changes and modifications that will beapparent to those familiar with the field of the invention are also partof the invention.

Aspects of the invention described with the term “comprising” should beunderstood to include the elements explicitly listed, and optionally,additional elements. Aspects of the invention described with “a” or “an”should be understood to include “one or more” unless the context clearlyrequires a narrower meaning.

Moreover, features of the invention described herein can be re-combinedinto additional embodiments that also are intended as aspects of theinvention, irrespective of whether the combination of features isspecifically mentioned above as an aspect or embodiment of theinvention. Also, only those limitations that are described herein ascritical to the invention should be viewed as such; variations of theinvention lacking features that have not been described herein ascritical are intended as aspects of the invention.

With respect to aspects of the invention that have been described as aset or genus, every individual member of the set or genus is intended,individually, as an aspect of the invention, even if, for brevity, everyindividual member has not been specifically mentioned herein. Whenaspects of the invention that are described herein as being selectedfrom a genus, it should be understood that the selection can includemixtures of two or more members of the genus. Similarly, with respect toaspects of the invention that have been described as a range, such as arange of values, every sub-range within the range is considered anaspect of the invention.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations specifically described herein. Although theapplicant(s) invented the full scope of the claims appended hereto, theclaims appended hereto are not intended to encompass within their scopethe prior art work of others. Therefore, in the event that statutoryprior art within the scope of a claim is brought to the attention of theapplicants by a Patent Office or other entity or individual, theapplicant(s) reserve the right to exercise amendment rights underapplicable patent laws to redefine the subject matter of such a claim tospecifically exclude such statutory prior art or obvious variations ofstatutory prior art from the scope of such a claim. Variations of theinvention defined by such amended claims also are intended as aspects ofthe invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Representative whole mount corneal immunofluorescencemicrographs showing lymphatics (CD31^(lo)/LYVE-1^(hi)) in normal and dryeye (DE) at day 14 (20× magnification).

FIG. 2: Representative whole mount corneal immunofluorescencemicrographs showing lymphatics (CD31^(lo)/LYVE-1^(hi)) in normal and dryeye (DE) at days 6, 10 and 14 (100× magnification).

FIG. 3a and FIG. 3b : Quantification of lymphatics in dry eye (DE)corneas. Morphometric analysis of corneal lymphangiogenesis in normaland DE days 6, 10 and 14 (100× magnification). Morphometric evaluationshowed significant increase in lymphatic area (LA) in dry eye comparedto normal corneas (FIG. 3a ). Significant increase in lymphatic caliber(LC) in dry eye compared to normal corneas was noticed only at day 14(FIG. 3b ). Data from a representative experiment of three performed isshown as mean±S.E.M and each group consists of four to five mice.

FIG. 4: Analysis of lymphangiogenic-specific growth factors. Real-timePCR analysis showing transcript levels of VEGF-A, VEGF-C and VEGF-D inthe dry eye corneas at different time points. A significant increase inVEGF-D was seen at day 6 whereas VEGF-A and VEGF-C increasedsignificantly only by day 14. Data from a representative experiment ofthree performed is shown as mean±S.E.M and each group consists of fourto five mice.

FIG. 5: Analysis of lymphangiogenic-specific growth factor receptors.Real-time PCR analysis showing transcript levels of VEGFR-2 and VEGFR-3in the dry eye corneas at different time points. Significant increase inVEGFR-3 was seen earliest at day 6 but VEGFR-2 increased significantlylater in disease at day 14. Data from a representative experiment ofthree performed is shown as mean±S.E.M and each group consists of fourto five mice.

FIG. 6: Enuneration of corneal CD11b⁺/LYVE-1⁺ cells. A significantincrease in the number of both CD11b⁺ and double stainedCD11^(hi)/LYVE-1⁺ cells in the dry eye corneas as compared to normal.Data from a representative experiment of three performed is shown asmean±S.E.M and each group consists of four to five mice.

FIG. 7: Increased homing of mature MTIC-II+CD11b+APC in the draining LNof DED mice. Flow cytometric analysis of draining lymph nodes showingsignificant increase in the frequencies of mature MHC-II⁺ CD11b⁺ APC inDED mice compared with normal mice. Data from a representativeexperiment of two performed is shown and each group consists of threemice.

FIG. 8: Analysis of lymphangiogenic-specific growth factors and theirreceptors. Real-time PCR analysis showing transcript levels of VEGF-A,VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in the dry eye corneas.

FIG. 9: Analysis of proinflammatory cytokines in conjunctiva. Real-timePCR analysis showing expression of cytokines IL-1α, IL-1β, IL-6, IL-17.The levels of all four cytokines in the conjunctiva showed significantlydecreased expression in anti-VEGF-C treated DED mice as compared tothose of untreated DED mice

FIG. 10: Analysis of inflammatory cytokines in draining lymph nodes.Real-time PCR analysis for IL-17 (Th17 cells) and IFN-γ (Th1 cells).Draining lymph nodes of anti-VEGF-C treated DED mice showedsignificantly decreased induction of T-cell mediated autoimmune responsecompared untreated DED mice.

FIG. 11: Enumeration of CD11b⁺ cells in DED corneas. Treatment withanti-VEGF-C antibodies significantly decreased infiltration of CD11b⁺cells (30%) in the DED corneas (day 14).

FIG. 12: Epifluorescent microscopic image of corneal wholemountsimmunostained with CD31 and LYVE-1.

FIG. 13: Quantification of number of infiltrating CD11b+ cells per mm²of cornea.

FIG. 14: In vivo blockade of VEGF-C ameliorates clinical signs of DED.Corneal fluorescein staining (CFS) score is used as readout for theclinical signs of dry eye inflammation, in anti-VEGF-C Ab-treated anduntreated mice. CFS scores were significantly decreased in the grouptreated with anti-VEGF-C antibody at days 5, 9 and 13 vs the untreatedgroup. Data shown as mean±S.E.M and each group consisted of 3-4 mice.

DETAILED DESCRIPTION Dry Eye Disease

Keratoconjunctivitis sicca (KCS), also called keratitis sicca, siccasyndrome, xerophthalmia, dry eye syndrome (DES), dry eye disease (DED),or simply dry eyes, is an eye disease caused by decreased tearproduction or increased tear film evaporation commonly found in humansand some animals. Typical symptoms of keratoconjunctivitis are dryness,burning and a sandygritty eye irritation that gets worse as the day goeson.

In the context of the present invention the term “dry eye condition”denotes any condition or syndrome which results in the manifestation ofdry eye symptoms. It includes an already existing condition as well aspseudo dry eye conditions, i.e. conditions high predisposition ofdeveloping dry eye syndromes. Dry eye disease may be as a result ofanother underlying condition causing dry eye, for example, Sjogren'ssyndrome, menopause or rheumatoid arthritis. Dry eye may also be acomplication of inflammation, e.g. Blepharitis or of a foreign body inthe eye. Dry eye may also be the result of infection, or a side effectof medications, or exposure to toxins, chemicals, or other substancesmay cause a symptom or condition of dry eye. Dry eye conditions may bemanifested by one or more ophthalmologic clinical symptoms as known inthe art. Examples of dry eye symptoms include, but are not limited to,foreign body sensation, burning, itching, irritation, redness, eye pain,blurred vision and/or degraded vision.

Keratoconjunctivitis sicca is characterized by inadequate tear filmprotection of the cornea because of either inadequate tear production orabnormal tear film constitution, which results in excessively fastevaporation or premature destruction of the tear film. The tear film isconstituted by 3 layers: (1) a lipid layer, produced by the Meibomianglands; (2) an aqueous layer, produced by the main and accessorylacrimal glands; and (3) a hydrophilic mucin layer, produced by theconjunctival goblet cells. Any abnormality of 1 of the 3 layers producesan unstable tear film and symptoms of keratitis sicca.

Sjogren's syndrome and autoimmune diseases associated with Sjogren'ssyndrome are also conditions associated with aqueous tear deficiency.Drugs such as isotretinoin, sedatives, diuretics, tricyclicantidepressants, antihypertensives, oral contraceptives, antihistamines,nasal decongestants, beta-blockers, phenothiazines, atropine, and painrelieving opiates such as morphine can cause or worsen this condition.Infiltration of the lacrimal glands by sarcoidosis or tumors, orpostradiation fibrosis of the lacrimal glands can also cause thiscondition.

Keratoconjunctivitis sicca can also be caused by abnormal tearcomposition resulting in rapid evaporation or premature destruction ofthe tears. When caused by rapid evaporation, it is termed evaporativedry eyes. In this, although the tear gland produces a sufficient amountof tears, the rate of evaporation of the tears is too rapid. There is aloss of water from the tears that results in tears that are too “salty”or hypertonic. As a result, the entire conjunctiva and cornea cannot bekept covered with a complete layer of tears during certain activities orin certain environments.

Aging is one of the most common causes of dry eyes. About half of allpeople who wear contact lenses complain of dry eyes. There are twopotential connections between contact lens usage and dry eye.Traditionally, it has been believed that soft contact lenses, whichfloat on the tear film that covers the cornea, absorb the tears in theeyes. However, it is also now known that contact lens usage damagescorneal nerve sensitivity, which may lead to decreased lacrimal glandtear production and dry eye. The effect of contact lenses on cornealnerve sensitivity is well established for hard contact lenses as well assoft and rigid gas permeable contact lenses. The connection between thisloss in nerve sensitivity and tear production is the subject of currentresearch. Dry eyes also occur or get worse after LASIK and otherrefractive surgeries. The corneal nerves stimulate tear secretion. Dryeyes caused by these procedures usually resolves after several months.Persons who are thinking about refractive surgery should consider this.

A variety of approaches can be taken to treat dry eyes. These can besummarized as: avoidance of exacerbating factors, tear stimulation andsupplementation, increasing tear retention, eyelid cleansing andtreatment of eye inflammation. Application of artificial tears every fewhours can provide temporary relief. Inflammation occurring in responseto tears film hypertonicity can be suppressed by mild topical steroidsor with topical immunosuppressants such as cyclosporine. Consumption ofdark-fleshed fish containing dietary omega-3 fatty acids is associatedwith a decreased incidence of dry eyes syndrome in women. Earlyexperimental work on omega-3 has shown promising results when used in atopical application (Rashid S et al (2008). Arch Ophthalmol 126 (2):219-225).

DED is increasingly recognized as an immune-mediated disorder.Desiccating stress in DED initiates an immune-based inflammationresponse that is sustained by the ongoing interplay between the ocularsurface and various pathogenic immune cells, primarily the CD4+ cells inthe conjunctivia and the CD11b+ monocytic cells in the corneal.Dessiccating stress induces secretion of inflammatory cytokines,especially IL-1, TNF-α and IL-6 by ocular tissues, which facilitates theactivation and migration of resident antigen presenting cells (APCs)toward the regional draining lymph nodes (LNs). In the LNs, these APCsstimulate naive T-cells, leading to the expansion of IL-17 secretingTh17 cells and interferon (IFN)-y-secreting Th1 cells. Once theseeffectors are generated in the LNs, they migrate to the ocular surfaceand secrete effector cytokines.

VEGF Family of Growth Factors

VEGF (Vascular Endothelial Growth Factor) is a sub-family of growthfactors, specifically the platelet-derived growth factor family ofcystine-knot growth factors. They are important signaling proteinsinvolved in both vasculogenesis (the de novo formation of the embryoniccirculatory system) and angiogenesis (the growth of blood vessels frompre-existing vasculature). Members of the platelet-derived growth factorfamily include the Placenta growth factor (PIGF), VEGF-A (also known asVEGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E.

VEGF-A, VEGF-C and VEGF-D exert their effects by variously binding toand activating two structurally related membrane receptor tyrosinekinases, VEGF receptor-1 (VEGFR-1 or Flt-I), VEGFR-2 (flk-1 or KDR), andVEGFR-3 (Flt-4). Members of the VEGF family may also interact with thestructurally distinct receptor neuropilin-1. Binding of a VEGF to thesereceptors initiates a signaling cascade, resulting in effects on geneexpression and cell survival, proliferation, and migration.

VEGF-A binds to VEGFR-1 (Flt-1) and to VEGFR-2 (KDR/Flk-1). VEGFR-2appears to mediate almost all of the known cellular responses to VEGF-A.The function of VEGFR-1 is less well-defined, although it is thought tomodulate VEGFR-2 signaling. VEGF-A is believed to play a central role inthe development of new blood vessels (angiogenesis) and the survival ofimmature blood vessels (vascular maintenance). VEGF-C and VEGF-D areligands for VEGFR-2 and VEGFR-3 and are involved in the mediation oflymphangiogenesis.

Lymphangiogenesis

Lymphangiogenesis refers to formation of lymphatic vessels, particularlyfrom pre-existing lymphatic vessels, but as used herein, the termapplies to formation of lymph vessels under any condition. It alsoapplies to the enlargement of lymphatic vessels, commonly known aslymphatic hyperplasia. Lymphangiogenesis plays an importantphysiological role in homeostasis, metabolism and immunity. Lymphaticvessel formation has also been implicated in a number of pathologicalconditions including neoplasm metastasis, oedema, rheumatoid arthritis,psoriasis and impaired wound healing.

The normal human cornea is avascular, thus suppressing the afferentlymphatic and efferent vascular arms or the immune cycle Inflammationhowever negates this immune and angiogenic privileged state of thecornea and giving the corneal and ocular surface the potential to mountan immune response. Our results show that corneal lymphatics play animportant role in mediating the corneal inflammation in dry eyes.Inhibition of corneal lymphangiogenesis decreases ocular surfaceinflammation in a well characterized mouse model of DED.

Lymphangiogenesis is regulated to a large extent by VEGF-C and VEGF-D.Lymphangiogenesis appears to be regulated by signaling mediated byVEGFR-3, particularly upon specifically binding its ligands, VEGF-C andVEGF-D.

During embryogenesis, lymphatic endothelial cell sprouting,proliferation and survival is promoted by VEGF-C. Lymphatic vessels failto develop in mice in which VEGF-C is absent (Vegfc knockout mice), andsuch mice develop severe edema. Indeed, absence of VEGF-C is embryoniclethal. Lymphatic vessel hypoplasia and lymphedema is exhibited in theskin of mice hemizygous for Vegfc (i.e. mice possessing one functionalallele).

Embryonic lymphangiogenesis is also partly regulated by VEGF-D, similarto VEGF-C. However, lymphangiogenesis during embryonic development isnot dependent upon VEGF-D, as demonstrated by Vegfd knockout mice. Thelymphatic system in Vegfd knockout mice is relatively normal and Vegfdknockout mice are viable and fertile. The absolute abundance oflymphatic vessels in the lung is, however, reduced by approximately 30%compared to wild-type mice.

Lymphatic vessels express VEGFR-3, the receptor for VEGF-C and VEGF-D,and both VEGF-C and VEGF-D signal predominantly through VEGFR-3. It isalso becoming apparent that lymphatic vessels variously express VEGFR-2.VEGF-C and VEGF-D are synthesized as prepro-polypeptides and areproteolytically processed by proprotein convertases. In humans, matureproteolytically processed forms of VEGF-C and VEGF-D bind to VEGFR-2 andVEGFR-3. In mice, mature VEGF-D binding is restricted to VEGFR-3.

VEGF-C and VEGF-D exist as homodimers, and it has been suggested thatthey may exist as VEGF-C-VEGF-D heterodimers. In addition to lymphaticvessels, VEGFR-3 is also expressed on blood vessel endothelial cellsduring development, thereby accounting for the severe vasculogenic andangiogenic defects observed during early embryogenesis in modelscomprising inactive VEGFR-3 signaling. The lymphatic system possessesalmost exclusive expression of VEGFR-3 in healthy tissues in adulthood,because VEGFR-3 expression in blood vessels declines following birth andduring adolescence. Thus, only lymphangiogenesis is inhibited in adultsby inhibition of the VEGF-C-VEGF-D-VEGFR-3 signaling axis.

Lymphatic vessels express neuropilin-2 (NRP-2), which can bind VEGF-C orVEGF-D. In lymphangiogenesis, NRP-2 is thought to act as a co-receptorto increase the binding affinity of VEGF-C or VEGF-D to VEGFR-3. NRP-2is required for lymphangiogenesis. Proliferation of lymphatic vesselendothelial cells was reduced and lymphatic vessels and capillariesfailed to develop in Nrp2 knockout mice in which NRP-2 is absent.Similarly, NRP-1 is capable of binding VEGF-C and VEGF-D.

Defective lymphatic capillaries are the underlying cause of Milroydisease and other rare hereditary forms of lymphedema in humans.Tyrosine kinase-inactivating point mutations of the VEGFR-3 gene havebeen identified as a major cause of Milroy disease, and VEGF-C andVEGF-D therapy has shown promising efficacy in preclinical animalmodels. However, previous work has only demonstrated lymphatic capillaryreconstitution, whereas effects on the collecting lymphatic vessels thatare more commonly damaged in lymphedema have not been addressed.

Lymphatic vessel growth in adult tissues can be induced byAngiopoietin-1 (ANG-1) through its binding to the tunica internaendothelial cell kinase receptor 2 (TIE-2 or TEK). Lymphatic vesselsprouting that was induced by ANG-1 was inhibited by an inhibitor ofVEGFR-3. Furthermore, VEGFR-3 was up-regulated by ANG-1 binding toTIE-2. TIE-2 expressed on lymphatic vascular endothelial cells may alsobe agonized by ANG-2 and ANG-3.

VEGF-C and VEGF-D may act as ligands for integrins. Specifically, VEGF-Cand VEGF-D have been shown to act as ligands for integrin α9β1. Celladherence and cell migration were promoted by each of VEGF-C and VEGF-Din cells expressing integrin α9β1. The effect could be blocked by ananti-integrin α9β1 antibody or siRNA directed to integrin α9β1.

Thus, in lymphangiogenesis, VEGFR-3 appears to be central. VEGFR-3specifically binds and is activated by ligands VEGF-C and VEGF-D. VEGF-Cand VEGF-D are synthesized as prepro-polypeptides and are activated byproteolytic processing by proprotein convertases. VEGF-C and VEGF-D alsobind specifically to NRP-2, which is thought to be a co-receptor forVEGFR-3. Both lymphangiogenesis and VEGFR-3 are up-regulated when ANG-1specifically binds to TIE-2. It is thought that binding of VEGF-C orVEGF-D to integrins, particularly integrin α9β1, also performs a role inlymphangiogenesis.

Lymphangiogenesis is mediated primarily by the interaction of growthfactors VEGF-C and VEGF-D on VEGFR-2 and VEGFR-3, and in particular onVEGFR-3. VEGF-A also contributes, albeit indirectly, tolymphangiogenesis by recruiting VEGF-C and VEGF-D secreting macrophages.Inhibition of VEGF-C and VEGF-D signaling pathways would thus constitutea new approach to the treatment of DED. The invention is however notrestricted to the inhibition of VEGF-C and VEGF-D signaling pathways andaccording to the present invention, other anti-lymphangiogenic agentscan be used to reduce the signs and symptoms of DED.

Anti-Lymphangiogenic Agents

Persons skilled in the art will appreciate from the foregoing thatinhibition of lymphangiogenesis can occur at a variety of biologicalpoints comprising any one or more of the interactions described. Forexample, inhibition may occur by targeting VEGF-D, VEGF-C or VEGFR-3.

An “anti-lymphangiogenic agent” as described herein refers to anysubstance that partially or fully blocks, neutralizes, reduces, inhibitsor antagonizes a biological activity of a molecular component ofsignaling mediated by VEGFR-3 or lymphangiogenesis. Alternatively, ananti-lymphangiogenic agent is any substance that partially or fullyblocks, neutralizes, reduces, inhibits or antagonizes a VEGF-C or VEGF-Dbiological activity. Thus, “inhibition” is the corresponding stateelicited by an inhibitor. A molecular component of signaling mediated byVEGFR-3 or lymphangiogenesis includes VEGFR-3, VEGFR-2, VEGF-C, VEGF-D,proprotein convertases, neuropilin-1 (NRP-1), neuropilin-2 (NRP-2),angiopoietin-1 (ANG-1), tunica interna endothelial cell kinase receptor(TIE-2) or integrin α9β1.

It is envisaged that practice of the invention extends to any inhibitorknown now or in the future.

Suitable classes of inhibitor molecules that target VEGF-C or VEGF-D orsignaling mediated by VEGFR-3, or lymphangiogenesis include antibodies,polypeptides, peptides, peptide mimetics, nucleic acid molecules, andsmall molecules. Such classes of inhibitor molecules are suitable alsofor inhibiting binding of ligands, for example VEGF-C or VEGF-D, tointegrins, particularly integrin α9β1.

Suitable VEGF-C, VEGF-D, VEGFR-3-mediated signaling or lymphangiogenesisantibody inhibitors include antagonist and neutralizing antibodies orantibody fragments.

Polypeptide, peptide, or peptide mimetic VEGF-C or VEGF-D inhibitors,VEGFR-3-mediated signaling inhibitors or lymphangiogenesis inhibitorsinclude fragments or amino acid sequence variants of native polypeptideor peptide components of VEGF-C, VEGF-D, VEGFR-3-mediated signaling orlymphangiogenesis.

Nucleic acid molecule inhibitors of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis include antisensemolecules, nucleic acids in triple-helix formation, small interferingRNA (siRNA), and ribozymes.

Small molecule inhibitors of VEGF-C or VEGF-D, VEGFR-3-mediatedsignaling or lymphangiogenesis include organic and inorganic molecules.

Inhibitors of VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis according to the present invention may exert theireffects by interacting with any one or more of VEGFR-3, VEGFR-2, VEGF-C,VEGF-D, proprotein convertases, NRP-1, NRP-2, ANG-1, TIE-2 or integrins,particularly integrin α9β1, in their DNA, RNA or polypeptide forms.

Inhibition of VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis according to the present invention may occur viainhibition of ligand availability for receptor binding, inhibition ofreceptor availability for ligand binding, inhibition of receptortyrosine kinase activity, or inhibition of co-receptor interaction.

As used herein, “availability” refers to the potential or actual amountof a molecule that performs some function in VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis and is present in abiological system. Availability may be relative or absolute. Forexample, if all copies of a gene encoding a polypeptide involved inlymphangiogenesis were rendered non-functional by genetic mutation andno functioning polypeptide was synthesized, then there would be noavailability of the polypeptide in an absolute sense. Alternatively, ifthe same gene was present with one functioning copy and 50% of thepolypeptide was synthesized, there would be reduced or inhibitedavailability in a relative sense. Similarly, other mechanisms may beenvisaged where availability is affected. Receptors may be transcribedor translated to a lesser degree when compared with a control, or thereceptor may be targeted by an antibody that binds specifically to theligand binding site, thereby reducing or inhibiting receptoravailability for ligand binding. Analogously, if ligand synthesis istargeted by an antisense inhibitor, or if an antibody inhibitor orsoluble receptor inhibitor specifically binds to the ligand, then therewill be reduction or inhibition of ligand availability for receptorbinding.

The term “specific binding” or “specifically binds” or “specific for”refers to binding where a molecule binds to a particular polypeptide orepitope on a particular polypeptide without substantially binding to anyother polypeptide or polypeptide epitope. Such binding is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target, for example, an excess of non-labeled target. Inthis case, specific binding is indicated if the binding of the labeledtarget to a probe is competitively inhibited by excess unlabeled target.As used herein, specific binding is used in relation to the interactionbetween the molecular components of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis. Specific binding isalso used in relation to the interaction between the molecularcomponents of VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis and agents that partially or fully block, neutralize,reduce or antagonize a biological activity of a molecule thatfacilitates VEGFR-3-mediated signaling or lymphangiogenesis. Specificbinding also applies to the interaction between the molecular componentsof VEGF-C or VEGF-D activity and agents that partially or fully block,neutralize, reduce or antagonize VEGF-C or VEGF-D biological activity.

In particular, specific binding refers to a molecule having a K_(d) atleast 2-fold less for the particular polypeptide or epitope on aparticular polypeptide than it does for a non-specific target.Preferably, specific binding refers to a molecule having a Kd at least4-fold, 6-fold, 8-fold or 10-foldless for the particular polypeptide orepitope on a particular polypeptide than it does for a non-specifictarget. Alternatively, specific binding can be expressed as a moleculehaving a Kd for the target of at least about 10⁻⁴ M, alternatively atleast about 10⁻⁵ M, alternatively at least about 10⁻⁶ M, alternativelyat least about 10⁻⁷ M, alternatively at least about 10⁻⁸ M,alternatively at least about 10⁻⁹ M, alternatively at least about 10⁻¹⁰M, alternatively at least about 10⁻¹¹ M, alternatively at least about10⁻¹² M, or less.

The person skilled in the art will appreciate that there exist manymechanisms for inhibiting VEGF-C or VEGF-D activity, VEGFR-3-mediatedsignaling or lymphangiogenesis. The principal aim is to reduce receptorsignaling. Some examples will be described below, but such a list is notintended to be limiting.

Antibody Inhibitors

The term “antibody” is used in the broadest sense and specificallycovers, for example, polyclonal antibodies, monoclonal antibodies(including antagonist and neutralizing antibodies), antibodycompositions with polyepitopic specificity, single chain antibodies, andfragments of antibodies, provided that they exhibit the desiredbiological or immunological activity.

An “antibody inhibitor” will specifically bind to a particularpolypeptide or epitope on a particular polypeptide without substantiallybinding to any other polypeptide or polypeptide epitope. Such bindingwill partially or fully block, neutralize, reduce or antagonize VEGF-Cor VEGF-D activity or a biological activity of a molecule thatfacilitates VEGFR-3-mediated signaling or lymphangiogenesis. Such targetmolecules include VEGFR-3, VEGFR-2, VEGF-C and VEGF-D, for example.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. Generally, the antibody will be purified (1)to greater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS PAGE under reducing or non-reducing conditions usingCoomassie blue or, preferably, silver stain. An isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred.

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (s.c.) or intraperitoneal (i.p.) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen (especially when synthetic peptides are used) to a protein thatis immunogenic in the species to be immunized. For example, the antigencan be conjugated to keyhole limpet hemocyanin (KLH), serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctionalor derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

In one protocol for generating polyclonal antibodies, animals areimmunized against the antigen, immunogenic conjugate, or derivative, bycombining the antigen, conjugate or derivative with 3 volumes ofFreund's complete adjuvant and injecting the solution intradermally atmultiple sites. One month later, the animals are boosted with 1/5 to1/10 the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater, the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Conjugates also can bemade in recombinant cell culture as protein fusions. Also, aggregatingagents such as alum are suitably used to enhance the immune response.

Monoclonal Antibodies

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method.

Monoclonal antibodies may be made using the hybridoma method in which amouse or other appropriate host animal, such as a hamster, is immunizedto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein used forimmunization. Alternatively, lymphocytes may be immunized in vitro.After immunization, lymphocytes are isolated and then fused with amyeloma cell line using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, which preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells(also referred to as fusion partner).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods. In addition,the hybridoma cells may be grown in vivo as ascites tumors in an animale.g., by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxyapatite chromatography, gelelectrophoresis, or dialysis.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures. The hybridoma cells serve as a preferredsource of such DNA. Once isolated, the DNA may be placed into expressionvectors, which are then transfected into host cells such as E. colicells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myelomacells that do not otherwise produce antibody protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells.

Monoclonal antibodies or antibody fragments can be isolated fromantibody phage libraries. High affinity (nM range) human antibodies canbe generated by chain shuffling, as well as combinatorial infection andin vivo recombination as a strategy for constructing very large phagelibraries. Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides. The monoclonal antibodies used hereininclude “chimeric” antibodies in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity.

Human and Humanized Antibodies

The anti-VEGF-C, anti-VEGF-D, anti-VEGFR-3-mediated signaling oranti-lymphangiogenesis antibodies used in the invention may comprisehumanized antibodies or human antibodies. Generally, a “humanizedantibody” is an antibody of non-human origin that has been modifiedusing recombinant DNA techniques to circumvent the problem of a human'simmune system reacting to an antibody as a foreign antigen. The standardprocedure of producing monoclonal antibodies produces mouse antibodies.Although murine antibodies are very similar to human ones, there aredifferences. Consequently, the human immune system recognizes mouseantibodies as foreign, rapidly removing them from circulation andcausing systemic inflammatory effects. “Humanized” forms of non-human(e.g., rodent) antibodies are chimeric antibodies that contain a reducedpercentage of sequence derived from the non-human antibody. Variousforms of humanized anti-VEGF-C, anti-VEGF-D, anti-VEGFR-3-mediatedsignaling or anti-lymphangiogenesis antibodies are contemplated.Humanized antibodies may be intact antibodies, such as intact IgG₁antibodies, antibody chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂, or other antigen-binding subsequences of antibodies). Humanizedantibodies include human antibodies (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman antibody are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human antibody and all or substantially all of the FR regionsare those of a human antibody consensus sequence. The humanized antibodyoptimally also will comprise at least a portion of an antibody constantregion (Fc), typically that of a human antibody.

Various humanization strategies have been described in the prior art andit is envisaged that practice of the invention extends to the use ofboth known humanization strategies and any new strategies to bedeveloped in the future. Examples of known humanization strategiesinclude those described by Studnicka (U.S. Pat. No. 5,869,619) andPadlan (1991, Molec. Immunol., 28, 489-498), Winter (U.S. Pat. No.5,225,539) and Jones et al (1986, Nature, 321, 522-525), Queen et al.(U.S. Pat. No. 5,693,761) and Foote (U.S. Pat. No. 6,881,557).

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production.

Alternatively, phage display technology can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors. Phage display canbe performed in a variety of formats. Several sources of V-gene segmentscan be used for phage display.

Antibody Fragments

“Antibody fragments” comprise a portion of an antibody, preferably theantigen binding or variable region of the intact antibody. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Antibodyfragments of particular interest are fragments that retainantigen-binding properties of the whole antibody, and are useful asinhibitors for practicing the invention.

Papain digestion of antibodies produces two identical antigen bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. Each Fabfragment is monovalent with respect to antigen binding, i.e., it has asingle antigen binding site. Pepsin treatment of an antibody yields asingle large F(ab′)₂ fragment which roughly corresponds to two disulfidelinked Fab fragments having divalent antigen binding activity and isstill capable of cross linking antigen. Fab′ fragments differ from Fabfragments by having additional residues at the carboxy terminus of theC_(H)1 domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a complete antigenrecognition binding site. This fragment consists of a dimer of one heavyand one light chain variable region domain in tight, non covalentassociation. From the folding of these two domains emanate sixhypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single chain Fv” abbreviated as “scFv” are antibody fragments thatcomprise the VH and VL antibody domains connected into a singlepolypeptide chain. Preferably, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding.

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance from the circulation.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies. However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage libraries.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments. According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues alsomay be used.

Other techniques for the production of antibody fragments will beapparent to the skilled practitioner. The antibody of choice is a singlechain Fv fragment (scFv). Fv and scFv are the only species with intactcombining sites that are devoid of constant regions; thus, they aresuitable for reduced nonspecific binding during in vivo use. Theantibody fragment may also be a “linear antibody”, which may bemonospecific or bispecific. The inhibitor also maybe a polypeptide orprotein comprising an antibody or antibody fragment linked to anotherentity to form a fusion protein.

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Bispecific antibodies can be preparedas full length antibodies or antibody fragments (e.g., F(ab′)₂bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities.

According to a different approach, antibody variable domains with thedesired binding specificity (antibody-antigen combining sites) are fusedto immunoglobulin constant domain sequences.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, one of theantibodies in the heteroconjugate can be coupled to avidin, the other tobiotin. Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents and cross-linkingtechniques are well known in the art.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Various techniques for making and isolating bispecificantibody fragments directly from recombinant cell culture have also beendescribed.

The term “diabodies” refers to small antibody fragments prepared byconstructing scFv fragments with short linkers (about 5 to 10 residues)between the VH and VL domains such that inter chain but not intra chainpairing of the V domains is achieved, resulting in a bivalent fragment,i.e., fragment having two antigen binding sites. Bispecific diabodiesare heterodimers of two “crossover” scFv fragments in which the VH andVL domains of the two antibodies are present on different polypeptidechains.

According to an alternative “diabody” technology for making bispecificantibody fragments, the fragments comprise a VH connected to a VL by alinker which is too short to allow pairing between the two domains onthe same chain. Accordingly, the VH and VL domains of one fragment areforced to pair with the complementary VL and VH domains of anotherfragment, thereby forming two antigen-binding sites. Another strategyfor making bispecific antibody fragments by the use of single-chain Fv(scFv) dimers has also been reported.

Antibodies with more than two valencies are contemplated for use in theinvention. For example, trispecific antibodies can be prepared.

Exemplary multivalent antibodies and binding constructs that aresuitable inhibitors for practicing the invention include those describedin International Patent Application No. PCT/US2005/007742 (published asWO 2005/087812 on 22 Sep. 2005) or US Patent Publication No.2005/0282233, filed Mar. 7, 2005, published Dec. 22, 2005, bothincorporated herein by reference in their entirety. These documentsdescribe, for example, an antibody substance that specifically binds tofirst and second growth factors selected from the group consisting ofhuman vascular endothelial growth factor-A (VEGF-A), human vascularendothelial growth factor-B (VEGF-B), human vascular endothelial growthfactor-C(VEGF-C), human vascular endothelial growth factor-D (VEGF-D),human vascular endothelial growth factor-E (VEGF-E), human placentalgrowth factor (PlGF), human platelet-derived growth factor-A (PDGF-A),human platelet-derived growth factor-B (PDGF-B), human platelet-derivedgrowth factor-C (PDGF-C), and human platelet-derived growth factor-D(PDGF-D), wherein each of said growth factors binds and stimulatesphosphorylation of at least one receptor tyrosine kinase, and whereinthe antibody substance inhibits the first and second growth factors towhich it binds from stimulating phosphorylation of said receptortyrosine kinases. Of particular interest for the present invention arebi-specific antibody substances that bind two of VEGF-C, VEGF-D, andVEGF-A, for example. These documents also describe an antibody substanceproduced by a process comprising: (a) screening a library of antibodymolecules to identify at least one antibody molecule that binds to afirst growth factor selected from the group consisting of human vascularendothelial growth factor-A (VEGF-A), human vascular endothelial growthfactor-B (VEGF-B), human vascular endothelial growth factor-C(VEGF-C),human vascular endothelial growth factor-D (VEGF-D), human vascularendothelial growth factor-E (VEGF-E), human placental growth factor(PlGF), human platelet-derived growth factor-A (PDGF-A), humanplatelet-derived growth factor-B (PDGF-B), human platelet-derived growthfactor-C(PDGF-C), and human platelet-derived growth factor-D (PDGF-D),wherein each of said growth factors binds and stimulates phosphorylationof at least one receptor tyrosine kinase; (b) screening molecule(s)identified in (a) to identify at least one molecule that binds to asecond growth factor selected from said group; (c) screening molecule(s)identified in step (b) to identify at least one molecule that inhibitsthe first and second growth factors to which it binds from stimulatingphosphorylation of said receptor tyrosine kinases, wherein the antibodysubstance comprises a molecule identified in step (C).

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. Antibodies that may be used in the present inventioncan be multivalent antibodies (which are other than of the IgM class)with three or more antigen binding sites (e.g. tetravalent antibodies),which can be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. A preferred dimerization domain comprises an Fc region ora hinge region. In this scenario, the antibody will comprise an Fcregion and three or more antigen binding sites amino-terminal to the Fcregion. A preferred multivalent antibody comprises three to about eight,but preferably four, antigen binding sites. The multivalent antibodycomprises at least one polypeptide chain (and preferably two polypeptidechains), wherein the polypeptide chain(s) comprise two or more variabledomains. For instance, the polypeptide chain(s) may compriseVD1-(X₁)_(n)-VD2-(X₂)_(n)-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X₁ and X₂ represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:V_(H)—C_(H)1-flexible linker-V_(H)—C_(H)1-Fc region chain; orV_(H)—C_(H)1-V_(H)-C_(H)1-Fc region chain. A multivalent antibodypreferably further comprises at least two (and preferably four) lightchain variable domain polypeptides. A multivalent antibody may, forinstance, comprise from about two to about eight light chain variabledomain polypeptides. The light chain variable domain polypeptidescontemplated here comprise a light chain variable domain and,optionally, further comprise a CL domain.

Peptide and Peptide Mimetic Inhibitors

In another embodiment, the inhibitor of VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis is a peptide or peptidemimetic. The peptide or peptide mimetic may reduce receptor availabilityfor native ligand binding.

As used herein, “peptide mimetic” and “peptidomimetic” are usedinterchangeably.

A peptide inhibitor is a peptide that binds specifically to a componentof VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis and inhibits or neutralizes the function of thatcomponent in the process of VEGF-C or VEGF-D activity, VEGFR-3-mediatedsignaling or lymphangiogenesis. Peptide inhibitors may be chemicallysynthesized using known peptide synthesis methodology or may be preparedand purified using recombinant technology. The preferred length ofpeptide inhibitors of VEGF-C or VEGF-D activity, VEGFR-3-mediatedsignaling or lymphangiogenesis is from about 6, 7, 8, 9 or 10 amino acidresidues to about 100 amino acid residues. It is contemplated thatlonger peptides may prove useful. Peptide inhibitors may be identifiedwithout undue experimentation using well known techniques. In thisregard, it is noted that techniques for screening peptide libraries forpeptides that are capable of specifically binding to a polypeptidetarget are well known in the art.

For any of the foregoing peptides, one preferred variation involvespeptides that have been modified to comprise an intramolecular bondbetween two non-adjacent amino acid residues of the primary sequence,thereby forming a cyclic peptide. For example, in one variation, thepeptide comprises a pair of cysteine residues, such as amino- andcarboxy-terminal cysteines, and the intramolecular bond comprises adisulfide bond between the cysteines. However, organic chemists andpeptide chemists are capable of synthesizing intramolecular bondsbetween a wide variety of amino acids using conventional techniques.

Exemplary peptidomimetic inhibitors of the VEGF-C and/or VEGF-Dsignaling pathways suitable for practicing the invention include thosedescribed in U.S. Pat. No. 7,045,133 (May 16, 2006), incorporated hereinby reference in its entirety. That patent describes, for example,monomeric monocyclic peptide inhibitors based on loop 1, 2 or 3 ofVEGF-D. A preferred peptide interferes with at least the activity ofVEGF-D and VEGF-C mediated by VEGF receptor-2 and VEGF receptor-3(VEGFR-3). A particularly preferred peptide interferes with the activityof VEGF-D, VEGF-C and VEGF mediated by VEGFR-2 and the activity ofVEGF-D and VEGF-C mediated by VEGFR-3. The patent also describes adimeric bicyclic peptide inhibitor which comprises two monomericmonocyclic peptides, each individually based on loop 1, 2 or 3 ofVEGF-D, linked together. Such dimeric bicyclic peptides may comprise twomonomeric monocyclic peptides which are the same or different. (See, forexample, Table 1-3 of the '133 patent.) Exemplary peptides in Table 1are reproduced below:

TABLE 1 Sequence and predicted and actual molecular masses (determinedby mass spectrometry) of peptides synthesized mass mass num- pre- actualber sequence dicted [M + H] 1

1358.52 1360 2

908.02 909.5 3

1116.37 1117.6 4

2793.18 2793.9 5

3566.09 3567.1 6

2019.6 2020.8 7

905.41 907 8

719.32 720.5 9

632.28 633.6 10

1014.5 1015.4 11

927.47 928.3

Additional peptide inhibitors useful for practicing this invention aredescribed in U.S. Pat. No. 7,611,711 (Nov. 3, 2009), incorporated hereinby reference in its entirety. This patent describes peptides that bindVEGFR-3 and inhibit VEGFR-3 ligands (VEGF-C and -D) from binding andstimulating the receptor. For example, in one embodiment, the inventionprovides an isolated peptide comprising the formula: X₁X₂X₃X₄X₅X₆X₇X₈(SEQ ID NO: 1), wherein X₁ through X₈ are amino acid residues, whereinthe peptide binds to VEGFR3, and wherein X₁ through X₈ are defined asfollows: the amino acid residue at X₁ is a glycine residue or aconservative substitution thereof; the amino acid residue at X₂ is atyrosine residue or a conservative substitution thereof; the amino acidresidue at X₃ is a tryptophan residue or a conservative substitutionthereof; the amino acid residue at X₄ is a leucine residue or aconservative substitution thereof; the amino acid residue at X₅ is athreonine residue or a conservative substitution thereof; the amino acidresidue at X₆ is an isoleucine residue or a conservative substitutionthereof; the amino acid residue at X₇ is a tryptophan residue or aconservative substitution thereof; and the amino acid residue at X₈ is aglycine residue or a conservative substitution thereof. Preferredpeptides are from 6 to 100 amino acids in length, e.g., 6, 7, 8, 9, 10,11, 12, . . . 97, 98, 99, or 100 amino acids in length. The peptides maybe made cyclic by the formation of at least one bond betweennon-adjacent amino acids. For example, in one variation, the peptidesare formed with terminal cysteines which can be made to form anintramolecular disulfide bond. Thus, in one preferred embodiment, thepeptide further comprises amino- and carboxy-terminal cysteine residues.For example, the peptide may comprise the amino acid sequence:CX₁X₂X₃X₄X₅X₆X₇X₈C (SEQ ID NO: 2), wherein X₁X₂X₃X₄X₅X₆X₇X₈ are definedas above, and C represents cysteine. In an alternative embodiment,additional residues are attached to X₁ or X₈, within the terminalcysteines.

Preferred conservative substitutions for these peptide molecules includeany of the following, in any combination: the conservative substitutionat position X₁ is selected from isoleucine, valine, leucine, alanine,cysteine, phenylalanine, proline, tryptophan, tyrosine, norleucine andmethionine; the conservative substitution at position X₂ is selectedfrom isoleucine, valine, leucine, alanine, cysteine, glycine,phenylalanine, proline, tryptophan, norleucine and methionine; theconservative substitution at position X₃ is selected from isoleucine,valine, leucine, alanine, cysteine, glycine, phenylalanine, proline,tyrosine, norleucine and methionine; the conservative substitution atposition X₄ is selected from isoleucine, valine, alanine, cysteine,glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine andmethionine; the conservative substitution at position X₅ is selectedfrom asparagine, glutamine, and serine; the conservative substitution atposition X₆ is selected from valine, leucine, alanine, cysteine,glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine ormethionine; the conservative substitution at position X₇ is selectedfrom isoleucine, valine, leucine, alanine, cysteine, glycine,phenylalanine, proline, tyrosine, norleucine and methionine; and theconservative substitution at position X₈ is selected from isoleucine,valine, leucine, alanine, cysteine, phenylalanine, proline, tryptophan,tyrosine, norleucine and methionine.

In on preferred embodiments, the invention provides an isolated peptidecomprising the sequence Y₁GYWLTIWGY₂ (SEQ ID NO: 3), wherein Y₁ areamino acids. In one variation, the peptide is made cyclic by a bondbetween Y₁ and Y₂. In a specific preferred embodiment, the peptidecomprises the sequence CGYWLTIWGC (SEQ ID NO: 4). Other specificexamples of peptides described in that patent include peptides thatcomprise any of the following sequences: SGYWWDTWF (SEQ ID NO: 15),SCYWRDTWF (SEQ ID NO: 16), KVGWSSPDW (SEQ ID NO: 17), FVGWTKVLG (SEQ IDNO: 18), YSSSMRWRH (SEQ ID NO: 19), RWRGNAYPG (SEQ ID NO: 20), SAVFRGRWL(SEQ ID NO: 21), WFSASLRFR (SEQ ID NO: 22), WQLGRNWI (SEQ ID NO: 23),VEVQITQE (SEQ ID NO: 24), AGKASSLW (SEQ ID NO: 25), RALDSALA (SEQ ID NO:26), YGFEAAW (SEQ ID NO: 27), YGFLWGM (SEQ ID NO: 28), SRWRILG (SEQ IDNO: 29), HKWQKRQ (SEQ ID NO: 30), MDPWGGW (SEQ ID NO: 31), RKVWDIR (SEQID NO: 32), VWDHGV (SEQ ID NO: 33), CWQLGRNWIC (SEQ ID NO: 34),CVEVQITQEC (SEQ ID NO: 35), CAGKASSLWC (SEQ ID NO: 36), CRALDSALAC (SEQID NO: 37), CYGFEAAWC (SEQ ID NO: 38), CYGFLWGMC (SEQ ID NO: 39),CSRWRILGC (SEQ ID NO: 40), CHKWQKRQC (SEQ ID NO: 41), CMDPWGGWC (SEQ IDNO: 42), CRKVWDIRC (SEQ ID NO: 43), CVWDHGVC (SEQ ID NO: 44), andCGQMCTVWCSSGC (SEQ ID NO: 45), and conservative substitution-analogsthereof, wherein the peptide binds human VEGFR-3. Preferred peptidescomprise these exact amino acid sequences, or sequences in which onlyone or only two conserved substitutions have been introduced. In anotherpreferred variation, the peptides are preferred with amino- andcarboxy-terminal cysteines, which permit formation of cyclic moleculesand dimers and multimers.

Nucleic Acid Molecules

Antisense Molecules

In yet another embodiment, the inhibitor of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis is an antisense moleculethat reduces transcription and/or translation of a component of VEGF-Cor VEGF-D activity, VEGFR-3-mediated signaling or lymphangiogenesis,thereby reducing VEGF-C or VEGF-D activity, VEGFR-3-mediated signalingor lymphangiogenesis.

The antisense molecule comprises RNA or DNA prepared using antisensetechnology, where, for example, an antisense RNA or DNA molecule acts toblock directly the translation of mRNA by hybridizing to targeted mRNAand preventing protein translation. Binding of antisense or senseoligonucleotides to target nucleic acid sequences results in theformation of duplexes that block transcription or translation of thetarget sequence by one of several means, including enhanced degradationof the duplexes, premature termination of transcription or translation,or by other means. The antisense oligonucleotides thus may be used toreduce or block expression of a component of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis, and thus VEGF-C orVEGF-D activity, VEGFR-3-mediated signaling or lymphangiogenesis. Sucholigonucleotides can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of componentsof VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis.

Inhibitors of VEGF-C or VEGF-D activity or signaling mediated byVEGFR-3, or lymphangiogenesis include antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences. Such a fragment generally comprises about 10 to40 nucleotides in length, preferably at least about 14 nucleotides,preferably from about 14 to 30 nucleotides.

Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones that are resistant toendogenous nucleases, or are covalently linked to other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, or intercalating agents to modify binding specificities of theantisense or sense oligonucleotide for the target nucleotide sequence.

Antisense materials and methods are further described below, e.g., inthe context of VEGFR-2 inhibitors. This discussion below also isapplicable to antisense materials and methods for other targets,including VEGFR-3, VEGF-C, and VEGF-D.

Small Interfering RNA (siRNA)

In one embodiment, it is envisaged that siRNA will inhibit VEGF-C orVEGF-D activity, VEGFR-3-mediated signaling or lymphangiogenesis.“siRNA” or “RNAi” are double-stranded RNA molecules, typically about 21nucleotides in length, that are homologous to a gene or polynucleotidethat encodes the target gene and interfere with the target gene'sexpression. Interfering RNA materials and methods are further describedbelow, e.g., in the context of VEGFR-2 inhibitors. This discussion belowalso is applicable to interfering RNA directed to other targets,including VEGFR-3, VEGF-C, and VEGF-D.

Nucleic Acid Molecules in Triple-Helix Formation

In another embodiment, the inhibitor of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis comprises nucleic acidmolecules in triple-helix formation. Nucleic acid molecules intriple-helix formation used to inhibit transcription should besingle-stranded and composed of deoxynucleotides. A DNA oligonucleotideis designed to be complementary to a region of the gene involved intranscription. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex.

Ribozymes

In a related embodiment, the inhibitor of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis is a ribozyme thatreduces transcription of a component of VEGF-C or VEGF-D activity orsignaling mediated by VEGFR-3, or a lymphangiogenic component.

A “ribozyme” is an enzymatic RNA molecule capable of catalyzing thespecific cleavage of RNA. Ribozymes act by sequence-specifichybridization to the complementary target RNA, followed byendonucleolytic cleavage. Specific ribozyme cleavage sites within apotential RNA target can be identified by known techniques.

Small Molecule Inhibitors

In a further embodiment, the inhibitor of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis is a small molecule.

A “small molecule” is defined herein to have a molecular weight belowabout 2000 daltons, and preferably below about 500 Daltons. Potentialinhibitors of VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis include small molecules that bind to the active site,the receptor binding site, or growth factor or other relevant bindingsite of components of VEGF-C or VEGF-D activity or VEGFR-3-mediatedsignaling, or lymphangiogenesis, thereby blocking the normal biologicalactivity of VEGF-C or VEGF-D, VEGFR-3-mediated signaling orlymphangiogenesis. Examples of small molecules include, but are notlimited to, synthetic non-peptidyl organic or inorganic compounds.

Small molecule inhibitors of VEGF-C or VEGF-D activity, VEGFR-3-mediatedsignaling or lymphangiogenesis may be identified without undueexperimentation using known techniques and chemically synthesized usingknown methodology. In this regard, it is noted that techniques forscreening organic molecule libraries for molecules that are capable ofbinding to a polypeptide target are known in the art.

Inhibition of Receptor Availability for Ligand Binding

Antibody Inhibitors

In one embodiment, the inhibitor of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis is an antibody. In apreferred embodiment, the inhibitor of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis is an anti-VEGFR-3antibody that reduces VEGFR-3 availability for ligand binding.

Suitable antibodies for use in the methods of the invention and meansfor their production are disclosed in WO2000/021560 and WO1995/021868and include a polyclonal or a monoclonal antibody that bindsspecifically to VEGFR-3 and blocks its signaling, a fragment of such anantibody, a chimeric antibody, a humanized antibody, and a bispecificantibody that binds specifically to VEGFR-3 and blocks its signaling andalso binds to another antigen.

In a preferred embodiment, the antibody inhibitor is a humanizedantibody. In another embodiment, the antibody inhibitor of VEGF-C orVEGF-D activity, VEGFR-3-mediated signaling or lymphangiogenesiscomprises a Fab, Fab′, or F(ab′)₂ fragment, or a single chain Fv (scFv)fragment.

Persons skilled in the art will appreciate that in particularembodiments, the monoclonal antibody may comprise antibody 9D9F9,disclosed in WO2000/021560 or 2E11D11 disclosed in WO2003/006104.Alternatively monoclonal antibodies that specifically bind to VEGFR-3and may be used according to the invention include antibodiesMM0003-7G63, RM0003-5F63, C28G5, KLT9, ZMD.251, mF4-31C1 and hF4-3C5. Aparticularly preferred monoclonal antibody is hF4-3C5, a fully-humanizedantagonist antibody to human VEGFR-3.

In an alternative embodiment, the inhibitor may comprise a bispecificantibody, particularly a diabody, that binds specifically to andneutralizes each of VEGFR-3 and a second target. One example of such adiabody is that derived from antibodies hF4-3C5 and IMC-1121, whichbinds specifically to and neutralizes each of VEGFR-3 and VEGFR-2.

An inhibitor of VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis according to the present invention also includes inone embodiment an antibody, as described above, that inhibits orneutralizes the receptor tyrosine kinase activity of VEGFR-3.

Peptide and Peptide Mimetic Inhibitors

The person skilled in the art will appreciate that particular inhibitorsof VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis that can be employed in a particular embodiment of thepresent invention are disclosed in WO2000/021560, WO2001/052875, andWO2002/057299, which are incorporated herein by reference. In oneembodiment, the inhibitor of VEGF-C or VEGF-D activity, VEGFR-3-mediatedsignaling or lymphangiogenesis comprises a peptide. Such a peptide to beused as an inhibitor of VEFC-C or VEGF-D activity, VEGFR-3-mediatedsignaling or lymphangiogenesis can be generated by random peptidesynthesis, by recombinant means from random oligonucleotides, or apeptide may be selected from a phage display library, according to thedisclosure of WO2002/057299 and WO2000/021560 and methods standard inthe art. Such a peptide can be identified with the aid of the VEGFR-3extracellular domain.

In a particular embodiment, the peptide inhibitor of VEGF-C or VEGF-Dactivity, VEGFR-3-mediated signaling or lymphangiogenesis comprises theamino acid sequence GYWX₁X₂X₃W (SEQ ID NO: 46), wherein X₁, X₂, and X₃comprise amino acids and wherein the peptide binds VEGFR-3, according toWO2002/057299. In a related embodiment, the peptide inhibitor comprisesthe amino acid sequence GYWX₁X₂X₃WX₄ (SEQ ID NO: 47), wherein X₄comprises an amino acid. In another embodiment, either of the precedingpeptides may further comprise an amino- and carboxy-terminus cysteineresidue. In a particular embodiment, the peptide comprises a cyclicpeptide. In an alternative embodiment, the peptide comprises a peptidedimer that binds to VEGFR-3, and in a preferred form, the peptidescomprising the dimer are the same, according to WO2002/057299.

In one embodiment, the peptidomimetic inhibitor is a monomericmonocyclic peptide inhibitor or dimeric bicyclic peptide inhibitor.Preferably, such peptidomimetic inhibitors are based on the peptidesequence of exposed loops of growth factor proteins, for example, loops1, 2, and 3 of VEGF-D. In a preferred embodiment, the peptidomimeticinhibitor comprises any one of: CASELGKSTNTFC (SEQ ID NO 5); CNEESLIC(SEQ ID NO: 6); or CISVPLTSVPC (SEQ ID NO: 7).

In one embodiment, the peptide mimetic inhibitor is prepared by themethods disclosed in WO2001/052875 and WO2002/057299. Peptides that maybe used as inhibitors of VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis are disclosed in WO2000/021560. Such peptides includea polypeptide comprising a fragment or analog of a vertebrate VEGF-Cpolypeptide, wherein the polypeptide and fragment or analog are capableof binding to VEGFR-3, but do not activate signaling, and a polypeptidecomprising a fragment or analog of a vertebrate VEGF-C or VEGF-Dpolypeptide, wherein the polypeptide and fragment or analog are capableof binding to VEGFR-3, but do not activate signaling.

The person skilled in the art will appreciate that inhibitors of VEGF-Dactivity, VEGFR-3-mediated signaling or lymphangiogenesis inhibitorsaccording to WO2002/057299 include peptides comprising the sequenceY₁GYWLTIWGY₂ (SEQ ID NO: 3), wherein Y, and Y₂ are amino acids. In onevariation, the peptide is made cyclic by a bond between Y and Y₂. In aspecific preferred embodiment, the peptide comprises the sequenceCGYWLTIWGC (SEQ ID NO: 4). Other peptide inhibitors comprise any of thefollowing amino acid sequences: SGYWWDTWF (SEQ ID NO: 15), SCYWRDTWF(SEQ ID NO: 16), KVGWSSPDW (SEQ ID NO: 17), FVGWTKVLG (SEQ ID NO: 18),YSSSMRWRH (SEQ ID NO: 19), RWRGNAYPG (SEQ ID NO: 20), SAVFRGRWL (SEQ IDNO: 21), WFSASLRFR (SEQ ID NO: 22), WQLGRNWI (SEQ ID NO: 23), VEVQITQE(SEQ ID NO: 24), AGKASSLW (SEQ ID NO: 25), RALDSALA (SEQ ID NO: 26),YGFEAAW (SEQ ID NO: 27), YGFLWGM (SEQ ID NO: 28), SRWRILG (SEQ ID NO:29), HKWQKRQ (SEQ ID NO: 30), MDPWGGW (SEQ ID NO: 31), RKVWDIR (SEQ IDNO: 32), VWDHGV (SEQ ID NO: 33), CWQLGRNWIC (SEQ ID NO: 34), CVEVQITQEC(SEQ ID NO: 35), CAGKASSLWC (SEQ ID NO: 36), CRALDSALAC (SEQ ID NO: 37),CYGFEAAWC (SEQ ID NO: 38), CYGFLWGMC (SEQ ID NO: 39), CSRWRILGC (SEQ IDNO: 40), CHKWQKRQC (SEQ ID NO: 41), CMDPWGGWC (SEQ ID NO: 42), CRKVWDIRC(SEQ ID NO: 43), CVWDHGVC (SEQ ID NO: 44), CGQMCTVWCSSGC (SEQ ID NO:45), or conservative substitutions-variants thereof. Preferred peptidescomprise these exact amino acid sequences, or sequences in which onlyone or only two conserved substitutions have been introduced. In anotherpreferred variation, the peptides comprise amino- and carboxy-terminalcysteines, which permit formation of cyclic molecules and dimers andmultimers. In yet another variation, peptide inhibitors include theamino acid sequence GYWX₁X₂X₃W (SEQ ID NO: 46), wherein X, X₂, and X₃comprise amino acids, the amino acid sequence GYWX₁XZX₃WX₄ (SEQ ID NO:47), wherein X₄ comprises an amino acid. In still another variation,these peptides further comprise amino- and carboxy-terminal cysteineresidues.

Nucleic Acid Inhibitors

In a preferred embodiment, the invention envisages use of a VEGFR-3antisense RNA, as disclosed in WO2000/021560, to inhibit the translationof VEGFR-3-encoding mRNA to eliminate or downregulate levels of VEGFR-3.Similarly, siRNA or nucleic acids in triple helix formation could beused to reduce VEGFR-3 availability for ligand binding.

Small Molecule Inhibitors

In a preferred embodiment, the small molecule is a small moleculeinhibitor of receptor tyrosine kinase activity. In a more preferredembodiment, the small molecule comprises PTK787/ZK22854, AZP2171, ZK991,KRN633, MAZ51, sorafenib, sunitinib (SU11248), axitinib (AG013736),vandetanib (ZD6474), or3-(indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione.

Inhibition of Ligand Availability for Receptor Binding

Antibody Inhibitors

According to one embodiment, inhibition of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis can be achieved usingantibodies that specifically bind and neutralize ligands for VEGFR-3,that is, VEGF-C and/or VEGF-D. Antibodies similar to anti-VEGFR-3antibodies described above are contemplated. Suitable antibodies andtheir means for production are disclosed in WO2000/021560. The personskilled in the art will appreciate that antibodies that bindspecifically to VEGF-D and may be used according to the inventioninclude monoclonal antibodies 2F8, 4A5 (also known as VD1), 4E10, 5F12,4H4 and 3C10 disclosed in WO2000/037025. A particularly preferredantibody is 4A5, and in particular, a humanized version thereof. Inanother embodiment, the chimeric or humanized antibody comprises SEQ IDNO: 56 and SEQ ID NO: 57, or the antibody comprises any one of SEQ IDNOs: 58 to 60 and any one of SEQ ID NOs: 61 to 63, as disclosed inWO2005/087177. Alternatively monoclonal antibodies that may be usedaccording to the invention include 28AT743.288.48, MM0007-7E79,RM0007-8C35, 78902, 78923, 78939, and 90409.

Similarly, monoclonal antibodies that bind VEGF-C may be employed. Theanti-VEGF-C antibodies will specifically bind to human VEGF-C or abiologically active fragment thereof, e.g. the mature fully-processedform. Such binding will partially or fully block, neutralize, reduce orantagonize VEGF-C activity. Suitable examples of such antibodies includeantibodies 103, MM0006-2E65 and 193208. Further examples of suchantibodies are found in U.S. Pat. No. 7,208,582 and U.S. Pat. No.7,109,308.

One example of an anti-VEGF-C antibody is a monoclonal antibody thatcompetitively inhibits the binding to VEGF-C of monoclonal anti-VEGF-Cantibody 69D09 produced by hybridoma ATCC PTA-4095 or having the heavyand light chain amino acid sequences as follows:

SEQ ID NO: 48 EVRLLESGGG  LVQPGGSLRL  SCAASGFTFR  PRAMAWVRQA  PGKGLEWVSS         10          20          30          40         50ISAQGASAYY  ADSVKGRFTI  SRDNSKNTLY  LQMNSLRAED  TAVYYCARDL         60          70          80          90          100SVSGFGPWGR  GTMVTVSSAS  TKGPSVFPLA  PSSKSTSGGT  AALGCLVKDY         110         120         130         140         150FPEPVTVSWN  SGALTSGVHT  FPAVLQSSGL  YSLSSVVTVP  SSSLGTQTYI          160         170         180         190         200CNVNHKPSNT  KVDKRVEPKS  CDKTHTCPPC  PAPELLGGPS  VFLFPPKPKD          210         220         230         240         250TLMISRTPEV  TCVVVDVSHE  DPEVKFNWYV  DGVEVHNAKT  KPREEQYNST         260         270         280         290         300YRVVSVLTVL  HQDWLNGKEY  KCKVSNKALP  APIEKTISKA  KGQPREPQVY          310         320         330         340         350TLPPSREEMT  KNQVSLTCLV  KGFYPSDIAV  EWESNGQPEN  NYKTTPPVLD          360         370         380         390         400SDGSFFLYSK  LTVDKSRWQQ  GNVFSCSVMH  EALHNHYTQK  SLSLSPGK          410         420         430         440       448

Sequence of Anti-VEGF-C Antibody Heavy Chain

SEQ ID NO: 49SYELTQPPSS  SGTPGQRVTI  SCSGSSSNIG  RHTVSWYQQV  PGTAPKLLIY          10          20          30          40         50SDDHRPSGVP  DRFSASKSGT  SASLTITGLQ  SEDEADYYCA  AWDDSLNGPW          60          70          80          90          100VFGGGTKLTV  LGQPKAAPSV  TLFPPSSEEL  QANKATLVCL  ISDFYPGAVT          110         120         130         140         150VAWKADSSPV  KAGVETTTPS  KQSNNKYAAS  SYLSLTPEQW  KSHRSYSCQV          160         170         180         190         200THEGSTVEKT  VAPTECS           210      217

Sequence of Anti-VEGF-C Antibody Light Chain

Another example of an anti-VEGF-C antibody is a monoclonal antibody thatbinds to the same epitope as the monoclonal anti-VEGF-C antibody 69D09produced by hybridoma ATCC PTA-4095 or a monoclonal antibody having theheavy and light chain amino acid sequences shown above. In oneembodiment, the anti-VEGF-C antibody is a fully-human anti-VEGF-Cmonoclonal antibody, including but not limited to 69D09 antibody orfragment thereof. The anti-VEGF-C antibody may be a humanized antibody.

Preferably, the anti-VEGF-C antibody is a human antibody produced bydeposited hybridoma ATC PTA-4095 (also referred to herein as “VGX-100”)or having the heavy and light chain amino acid sequences shown above.

Alternatively, antibodies may bind proprotein convertases, enzymesresponsible for processing VEGF-C and VEGF-D from their prepro-forms totheir activated forms, and reduce, inhibit or neutralize such activitythereby limiting the amount of proteolytically processed ligandavailable for binding to VEGFR-3. Again, antibodies corresponding withanti-VEGFR-3 antibodies described above are envisaged. Such antibodiesare disclosed in WO05/112971 and include neutralizing antibodies toinhibit the biological action of proprotein convertases.

Peptide Inhibitors

Inhibitors of VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis as used in the present invention include inhibitors ofproprotein convertases. As noted, one class of inhibitor of proproteinconvertases comprises antibodies. Another class of inhibitor ofproprotein convertases includes peptide inhibitors.

Peptide inhibitors of proprotein convertases are disclosed inWO05/112971 and include prosegments of proprotein convertases,inhibitory variants of anti-trypsin and peptidyl haloalkylketoneinhibitors.

Representative inhibitory prosegments of proprotein convertases includethe inhibitory prosegments of PC5A (also known as PC6A), PC5B (alsoknown as PC6B), PACE4, PC1 (also known as PC3), PC2, PC4, PC7 and Furin.A representative inhibitory variant of anti-trypsin is α-1 antitrypsinPortland, an engineered variant of naturally occurring antitrypsin thatinhibits multiple proprotein convertases. Representative peptidylhalomethyl ketone inhibitors includedecanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK),decanoyl-Phe-Ala-Lys-Arg-chloromethylketone (Dec-FAKR-CMK),decanoyl-Arg-Glu-Ile-Arg-chloromethylketone (Dec-REIR-CMK), anddecanoyl-Arg-Glu-Lys-Arg-chloromethylketone (Dec-REKR-CMK). Theseinhibitors of proprotein convertases, such as Dec-RVKR-CMK or theinhibitory prosegments of proprotein convertases, can be used to blockthe activation of VEGF-C and VEGF-D and thereby inhibit VEGF-C or VEGF-Dactivity, VEGFR-3-mediated signaling or lymphangiogenesis induced bypartially processed or fully processed VEGF-C or VEGF-D.

Soluble Receptors

According to another embodiment, VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis can be inhibited usingsoluble receptors that bind VEGFR-3 ligands. Soluble receptors capableof binding VEGF-C and VEGF-D, thereby inhibiting VEGF-C or VEGF-Dactivity or signaling via VEGFR-3, are disclosed in WO2000/023565,WO2000/021560, WO2002/060950 and WO2005/087808. Such inhibitors ofVEGF-D activity, VEGFR-3-mediated signaling or lymphangiogenesisinhibitors include soluble VEGFR-2, VEGFR-3, NRP-1, and NRP-2.

According to one embodiment, soluble receptor constructs useful forpracticing the present invention are described in International PatentApplication No. PCT/US02/01784, filed Jan. 22, 2002 (WO 2002/060950,published Aug. 8, 2002), incorporated here by reference in its entirety,and in International Patent Application No. PCT/US2005/007741, filedMar. 7, 2005 (WO 2005/087808, published Sep. 22, 2005), incorporatedhere by reference in its entirety. As described therein, the receptortyrosine kinases that bind the VEGF or PDGF family of ligands include anextracellular domain, a hydrophobic transmembrane domain, and anintracellular domain. The extracellular domain can be used as a ligandtrap by formulating it as a soluble formulation, optionally fused withan additional component, such as polyethylene glycol or an antibodyconstant region to improve serum half-life.

The complete ECD of PDGFRs and VEGFRs is not required for ligand (growthfactor) binding. The ECD of VEGFR-1 (R-1) and VEGFR-2 (R-2) consists ofseven Ig-like domains and the ECD of VEGFR-3 (R-3) has six intactIg-like domains—D5 of R-3 is cleaved post-translationally into disulfidelinked subunits leaving VEGFR-3. Veikkola, T., et al., Cancer Res.60:203-212 (2000). In general, receptor fragments of at least the firstthree Ig-like domains for this family are sufficient to bind ligand. ThePDGFRs have five Ig-like domains.

TABLE 1A Immunoglobulin-like domains for VEGFR-2 and VEGFR-3 R-2 R-2 R-3R-3 SEQ ID SEQ ID SEQ ID SEQ ID NO: 50 NO: 51 NO: 54 NO: 55 positionspositions positions positions D1 145-316  48-105 158-364  47-115 D2436-610 145-203 479-649 154-210 D3 724-931 241-310 761-961 248-314 D41039-1204 346-401 1070-1228 351-403 D5 1321-1600 440-533 1340-1633441-538 D6 1699-1936 566-645 1739-1990 574-657 D7 2050-2221 683-7402102-2275 695-752

Soluble receptor constructs for use as a ligand trap for VEGF-C or -Dpreferably comprise at least one Ig-like domain of a VEGFR as describedin Table 2, to as many as seven. The construct optionally will includesequence before the most N-terminally positioned Ig-like domain,optionally will include sequence beyond the most C-terminally Ig-likedomain, and optionally will include sequence between the Ig-domains aswell. Variants, e.g., with one or more amino acid substitutions,additions, or deletions of an amino acid residue, are also contemplated.Likewise, chimeras, e.g., combinations of Ig-like domains from differentreceptors, are contemplated. In some embodiments, the soluble receptorcomprises a receptor fragment comprising at least the first threeIg-like domains of a receptor tyrosine kinase.

In one embodiment, referring specifically to the VEGFR-3 sequence,soluble receptors are contemplated in which the soluble receptorpolypeptide comprises a portion of a mammalian, preferably human,VEGFR-3 extracellular domain (EC) wherein the portion binds to at leastone VEGFR-3 ligand and comprises at least the first, second and thirdIg-like domains of the VEGRF-3-EC, and wherein the polypeptide lacksVEGFR-3 Ig-like domains 4-7 and preferably any transmembrane domain.

In another embodiments, referring specifically to the VEGFR-3 sequence,soluble receptors are contemplated in which the soluble receptorpolypeptide comprises an amino acid sequence at least 90, 91, 92, 93,94, or 95% identical to a VEGFR-3 fragment, wherein the VEGFR-3 fragmentcomprises an amino acid sequence consisting of a portion of SEQ ID NO:53, wherein the carboxy-terminal residue of the fragment is selectedfrom the group consisting of positions 211 to 247 of SEQ ID NO: 53, andwherein the fragment and the polypeptide bind VEGF-C or VEGF-D. In somevariations, the fragment has an amino terminal amino acid selected fromthe group consisting of positions of 1 to 47 of SEQ ID NO: 53. In somevariations, the VEGFR-3 fragment used to make the soluble receptor hasan amino terminal residue selected from the group consisting ofpositions 1 to 47 of SEQ ID NO: 53, and a carboxy-terminal residueselected from the group consisting of positions 226 to 775 of SEQ ID NO:53, wherein VEGFR-3 fragment binds at least one of VEGF-C and VEGF-D.Specific peptides include a fragment of R-3 defined by positions 1-226,positions 1-229, and positions 1-329, positions 47-224, positions47-225, positions 47-226, positions 47-227, positions 47-228, positions47-229, positions 47-230, positions 47-231, positions 47-232, positions47-236, positions 47-240, positions 47-245, positions 47-314, positions47-210, and positions 47-247.

Soluble receptors that bind VEGF-C and -D can also be constructed from aVEGFR-2 amino acid sequence. Referring to the human VEGFR-2 sequence(SEQ ID NO: 51), exemplary R2 fragments for use in a soluble receptorhave an amino terminal residue selected from the group consisting ofpositions 1 to 118 of SEQ ID NO: 51, and a carboxy-terminal residueselected from the group consisting of positions 326 to 764 of SEQ ID NO:51, wherein VEGFR-2 fragment binds at least one of VEGF-C and VEGF-D(and may also bind VEGF-A or other VEGF's). In some variations, theamino terminal residue from the VEGFR-2 sequence is selected from thegroup consisting of positions 1 to 192 of SEQ ID NO: 51, and the carboxyterminal residue is selected from the group consisting of positions 393to 764 of SEQ ID NO: 51. In still other variations, the amino-terminalresidue is selected from the group consisting of positions 1 to 48 ofSEQ ID NO: 51, and a carboxy terminal residue selected from the groupconsisting of positions 214 to 764 of SEQ ID NO: 51. In someembodiments, the soluble receptor based on VEGFR-2 comprises a VEGFR-2sequence selected from positions 24-326, positions 118-326, positions118-220, positions 118-226, and positions 118-232, positions 106-240,positions 112-234, positions 114-220, positions 115-220, positions116-222, positions 117-220, positions 118-221, positions 118-222,positions 118-223, positions 118-224, positions 118-228, positions48-203, positions 145-310 and positions 48-310.

As described in WO 2005/087808, these or other exemplary solublereceptor constructs can be combined to make multivalent bindingconstructs. The receptor fragments, binding constructs, and otherpeptide molecules may be fused to heterologous peptides to confervarious properties, e.g., increased solubility, modulation of clearance,targeting to particular cell or tissue types. In some embodiments, thereceptor fragment is linked to a Fc domain of IgG or otherimmunoglobulin. In some embodiments, a receptor fragment is fused toalkaline phosphatase (AP). Methods for making Fc or AP fusion constructsare found in WO 02/060950.

Nucleic Acid Inhibitors

In another embodiment of the invention, antisense oligonucleotides areused as inhibitors of proprotein convertases. The antisenseoligonucleotides preferably inhibit expression of proprotein convertasesby inhibiting transcription or translation of proprotein convertases. Ina further embodiment, the antagonizing agent is small interfering RNAs(siRNA, also known as RNAi, RNA interference nucleic acids). Alsocontemplated are methods of inhibiting the target gene expression ortarget protein function utilizing ribozymes and triplex-forming nucleicacid molecules.

Similarly, in a related embodiment, antisense, siRNA and ribozymeinhibitors directed to VEGF-C and/or VEGF-D are included as inhibitorsof VEGF-C or VEGF-D activity, VEGFR-3-mediated signaling orlymphangiogenesis exerting their effects by reducing transcriptionand/or translation of VEGF-C and VEGF-D.

Peptide and Peptide Mimetic Inhibitors

According to one embodiment, the inhibitor to be used in the inventioncomprises a peptide that reduces the availability of ligand to bind toVEGFR-3. Such a peptide can be generated by random peptide synthesis, byrecombinant means from random oligonucleotides, or a peptide may beselected from a phage display library by methods standard in the art. Ina particular embodiment, the peptide will be derived from VEGFR-3 orVEGFR-2 and will bind specifically to VEGF-C or VEGF-D such that theligand available for binding to native VEGFR-3 is reduced. Such apeptide may be identified with the aid of the VEGF-C or VEGF-D.

Small Molecule Inhibitors

In one embodiment, the small molecule inhibitor is a small moleculeinhibitor of a proprotein convertase. In a particular embodiment, theproprotein convertase is furin and the small molecule comprises B3(CCG8294, naphthofluorescein disodium) or a derivative of2,5-dideoxystreptamine.

Tyrosine Kinase Inhibitors

In another embodiments, the anti-lymphangiogenic agent is a tyrosinekinase inhibitor that inhibits VEGFR-3 activity, which means aninhibitor of receptor tyrosine kinase activity that selectively ornon-selectively reduces the tyrosine kinase activity of a VEGFR-3receptor. Such an inhibitor generally reduces VEGFR-3 tyrosine kinaseactivity without significantly effecting the expression of VEGFR-3 andwithout effecting other VEGFR-3 activities such as ligand-bindingcapacity. A VEGFR-3 kinase inhibitor can be a molecule that directlybinds the VEGFR-3 catalytic domain, for example, an ATP analog. AVEGFR-3 kinase inhibitor can bind the VEGFR-3 catalytic domain throughone or more hydrogen bonds similar to those anchoring the adenine moietyof ATP to VEGFR-3 (Engh et al., J. Biol. Chem. 271:26157-26164 (1996);Tong et al., Nature Struc. Biol. 4:311-316 (1997); and Wilson et al.,Chem. Biol. 4:423-431 (1997)). A VEGFR-3 kinase inhibitor also can bindthe hydrophobic pocket adjacent to the adenine binding site (Mohamedi etal., EMBO J. 17:5896-5904 (1998); Tong et al., supra, 1997; and Wilsonet al., supra, 1997).

VEGFR-3 kinase inhibitors useful in the invention include specificVEGFR-3 kinase inhibitors such as indolinones that differentially blockVEGF-C and VEGF-D induced VEGFR-3 kinase activity compared to that ofVEGFR-2. Such specific VEGFR-3 kinase inhibitors, for example, MAE106and MAZ51 can be prepared as described in Kirkin et al., Eur. J.Biochem. 268:5530-5540 (2001). Additional VEGFR-3 kinase inhibitors,including specific, selective and non-selective inhibitors, are known inthe art or can be identified using one of a number of well known methodsfor assaying for receptor tyrosine kinase inhibition.

As an example, a VEGFR-3 kinase inhibitor can be identified using a wellknown ELISA assay to analyze production of phosphorylated tyrosine asdescribed, for example in Hennequin et al., J. Med. Chem. 42:5369-5389(1999) and Wedge et al., Cancer Res. 60:970-975 (2000). Such an assaycan be used to screen for molecules that inhibit VEGFR-3 in preferenceto other vascular endothelial growth factor receptors such as VEGFR-1and in preference to unrelated tyrosine kinases such as fibroblastgrowth factor receptorl (FGFR1). Briefly, molecules to be screened canbe incubated for 20 minutes at room temperature with a cytoplasmicreceptor domain in a HEPES (pH 7.5) buffered solution containing 10 mMMnCl₂ and 2 μM ATP in 96-well plates coated with a poly(Glu, Ala, Tyr)6:3:1 random copolymer substrate (SIGMA; St. Louis, Mo.). Phosphorylatedtyrosine can be detected by sequential incubation with mouse IgGanti-phosphotyrosine antibody (Upstate Biotechnology; Lake Placid,N.Y.), a horseradish peroxidase-linked sheep anti-mouse immunoglobulinantibody (Amersham; Piscataway, N.J.), and 2,2′azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Roche Molecular Biochemicals,Indianapolis, Ind.). In such an in vitro kinase assay, the source ofVEGFR-3 can be, for example, a lysate prepared from an insect cellinfected with recombinant baculovirus containing a cytoplasmic receptordomain, for example, encoding residues 798 to 1363 of human VEGFR-3.

The term VEGFR-3 kinase inhibitor, as used herein, encompasses specific,selective and non-selective inhibitors of VEGFR-3. A specific VEGFR-3kinase inhibitor reduces the tyrosine kinase activity of VEGFR-3 inpreference to the activity of most or all unrelated receptor tyrosinekinases such as FGFR1 and in preference to the activity of the vascularendothelial growth factor receptors, VEGFR-1 and VEGFR-2. A selectiveVEGFR-3 kinase inhibitor reduces the tyrosine kinase activity of VEGFR-3in preference to most or all unrelated receptor tyrosine kinases such asFGFR1. Such a selective VEGFR-3 inhibitor can have an IC₅₀ forinhibition of an isolated VEGFR-3 cytoplasmic domain that is, forexample, at least 10-fold less than the IC₅₀ for both VEGFR-1 andVEGFR-2. In particular embodiments, the invention provides a selectiveVEGFR-3 kinase inhibitor having an IC₅₀ for inhibition of an isolatedVEGFR-3 cytoplasmic domain that is at least 20-fold, 30-fold, 40-fold,50-fold, 100-fold, 200-fold, 300-fold, 400-fold or 500-fold less thanthe IC₅₀ for both VEGFR-1 and VEGFR-2. In contrast, a non-selectiveVEGFR-3 kinase inhibitor reduces the tyrosine kinase activity of VEGFR-1or VEGFR-2 or both to a similar extent as VEGFR-3.

Antibody Inhibitors Affecting Ligand—Receptor Complex

In one embodiment, the invention includes use of bispecific antibodies,as described above, as inhibitors of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis, specifically inhibitingligand-receptor complexes.

Suitable antibodies and their means for production are disclosed inWO2000/021560 and include a bispecific antibody that binds specificallyto an epitope or epitopes derived from a VEGFR-3-(VEGFR-3 ligand)complex (receptor-ligand complex) and blocks VEGFR-3 signaling.

Inhibition of Co-Receptor Interaction

Antibody Inhibitors Affecting Co-Receptors of VEGFR-3

In a further embodiment, inhibitors of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis include antibodies, asdescribed above, that bind specifically to and reduce, inhibit orneutralize co-receptor binding to VEGFR-3. Such antibodies may bedirected to a co-receptor, a ligand-co-receptor binary complex, aco-receptor-receptor binary complex, or a ligand-co-receptor-receptorternary complex. Co-receptors include NRP-1 and NRP-2. The personskilled in the art will understand that monoclonal antibodies thatspecifically bind NRP-1 or NRP-2 and may be used according to theinvention include antibodies 1B3, 3G6-2C5, AD5-17F6, 446915, 446921,130603, 130604, 96009, 3B8, 54, 257103, 257107, A-12, and C-9.Alternatively, a bispecific antibody which specifically binds to NRP-2receptor and a VEGF-C polypeptide, as disclosed in WO2003/029814, may beused according to the invention.

Peptide Inhibitors Affecting Co-Receptors of VEGFR-3

In another embodiment, a peptide inhibitor comprising a peptide dimermay target one or more receptors and/or co-receptors. Co-receptorsinclude NRP-1 and NRP-2. As disclosed in WO2002/057299, in a particularembodiment, the peptide dimer comprises one peptide that binds VEGFR-3and a second peptide that binds to any one of VEGFR-1, VEGFR-2, NRP-1,or NRP-2.

Small Molecule and Nucleic Acid Inhibitors Affecting Co-Receptors ofVEGFR-3

According to the present invention, it is also envisaged that smallmolecules, antisense molecules, siRNA and ribozymes, as described above,can be utilized as inhibitors of VEGF-D activity, VEGFR-3-mediatedsignaling or lymphangiogenesis by targeting co-receptors that interactwith VEGFR-3. Such co-receptors include NRP-1 and NRP-2.

Inhibition of Downstream Signaling

Alternatively, an inhibitor of VEGF-C or VEGF-D activity,VEGFR-3-mediated signaling or lymphangiogenesis according to any of theforegoing descriptions may disrupt downstream intracellular VEGFR-3signaling, as disclosed in WO2000/021560.

Therapeutic Uses of the Anti-Lymphangiogenic Agents

In yet another embodiment, the invention provides numerous methods ofusing the anti-lymphangiogenic agents described herein. Generallyspeaking, the anti-lymphangiogenic agents described herein are usefulfor inhibiting cellular processes that are mediated through signaltransduction through VEGFR-2 or VEGFR-3 for prophylaxis or therapy ofdry eye disease.

Thus, in one variation, described herein is a method of prophylaxis ortherapy for dry eye disease comprising administering to a subject inneed of prophylaxis or therapy for dry eye disease a compositioncomprising an anti-lymphangiogenic agent. Preferably, the amount of theanti-lymphangiogenic agent employed is effective to inhibit the bindingof VEGF-C and/or VEGF-D ligand to VEGFR-3 or the stimulatory effect ofVEGF-C and/or VEGF-D on VEGFR-3.

Dose response studies permit accurate determination of a proper quantityof anti-lymphangiogenic agent to employ. Effective quantities can beestimated, for example, from measurements of the binding affinity of apolypeptide for a target receptor, of the quantity of receptor presenton target cells, of the expected dilution volume (e.g., patient weightand blood volume for in vivo embodiments), and of polypeptide clearancerates. For example, existing literature regarding dosing of anti-VEGF-Cantibodies known also provides guidance for dosing of theanti-lymphangiogenic agents described herein.

The anti-lymphangiogenic agent described herein can be administeredpurely as a prophylactic treatment to prevent dry eye disease insubjects at risk for developing dry eye disease, or as a therapeutictreatment to subjects afflicted with dry eye disease, for the purpose ofinhibiting lymphangiogenesis in the eye of a subject in need thereof.

Subjects who are at risk for developing dry eye disease include subjectsconsidering or who have already undergone refractive surgery; subjectsover the age of sixty five; female subjects experiencing hormonalchanges brought on by pregnancy, lactation, oral contraceptives,menstruation and post-menopause; subjects afflicted with rheumatoidarthritis, diabetes, thyroid abnormalities, asthma, cataracts, glaucomaor lupus; subjects taking medication(s) that decrease the body's abilityto produce lubricating tears such as decongestants, antidepressants,antihistamines, blood pressure medication, oral contraceptives,diuretics, ulcer medication, tranquilizers and beta blockers; subjectsthat wear contact lenses; subjects exposed to environmental conditionsthat increase tear evaporation such as smoke, fluorescent light, airpollution, wind, heat, air conditioning and dry climates; and subjectsthat are heavy computer users (i.e., subjects that spend hours staringat computer displays that ignore their normal blinking process.

In one embodiment, described herein is a method of inhibiting dry eyedisease in a subject at risk for developing dry eye disease comprisingidentifying a subject as being at risk for developing dry eye diseaseand administering an anti-lymphangiogenic agent to the subject. Theamount of the anti-lymphangiogenic agent administered to the subject ispreferably in an amount effective to inhibit the development of dry eyedisease in the subject.

In another embodiment, described herein is a method of selecting atherapeutic regimen for a subject in need thereof comprising screening asubject for one or more symptoms of dry eye disease and prescribing forthe subject administration of a composition comprising ananti-lymphangiogenic agent described herein. In another embodiment,described herein is a method of treating a subject affected with dry eyedisease comprising identifying a subject as having one or more symptomsof dry eye disease and administering a composition comprising ananti-lymphangiogenic agent to the subject. Symptoms associated with dryeye disease include, but are not limited to, foreign body sensation,burning, itching, irritation, redness, eye pain, blurred vision,degraded vision, excessive tearing, dryness and a sandygritty eyeirritation that gets worse as the day goes on.

In some embodiments, the methods described herein do not includeadministering an anti-lymphangiogenic agent as described herein to asubject that has undergone corneal transplant surgery.

In some embodiments, the methods described herein further compriseprescribing (or administering) a standard of care regimen for thetreatment of dry eye disease. In the context of methods describedherein, “standard of care” refers to a treatment that is generallyaccepted by clinicians for a certain type of patient diagnosed with atype of illness. For dry eye disease, for example, an aspect of theinvention is to improve standard of care therapy with co-therapy withanti-lymphangiogenic agents described herein that inhibitlymphangiogenesis. Exemplary standard of care regimens for dry eyedisease include, but are not limited to, eyelid hygiene, topicalantibiotics (including, but not limited to erythromycin or bacitracinointments), oral tetracyclines (tetracycline, doxycycline, orminocycline), anti-inflammatory compounds (including, but not limitedto, cyclosporine) and corticosteroids.

Also contemplated are methods of treating a subject with dry eye diseasethat is hypo-responsive to a standard of care regimen for the treatmentof dry eye disease comprising administering an anti-lymphangiogenicagent to the subject in an amount effective to treat dry eye disease.

In a preferred embodiment, the mammalian subject is a human subject.Practice of methods of the invention in other mammalian subjects,especially mammals that are conventionally used as models fordemonstrating therapeutic efficacy in humans (e.g., primate, porcine,canine, or rabbit animals), is also contemplated.

Combination Therapy

Combination therapy embodiments of the invention include products andmethods. Exemplary combination products include two or more agentsformulated as a single composition or packaged together in separatecompositions, e.g., as a unit dose package or kit. Exemplary combinationmethods include prescribing for administration, or administration of twoor more agents simultaneously or in tandem.

A combination of an anti-lymphangiogenic agent with one or moreadditional therapeutic agents in the methods described herein may reducethe amount of either agent needed as a therapeutically effective dosage,and thereby reduce any negative side effects the agents may induce invivo. Combination therapy preferably results in improved efficiencycompared to either agent alone. Additional therapeutics or second agentscontemplated for use in combination with an anti-lymphangiogenic agentdescribed herein include a VEGFR-2 inhibitor product, a tyrosine kinaseinhibitor that inhibits VEGFR-2 and/or VEGFR-3 signaling, ananti-inflammatory agent; an immunosuppressive agent (e.g.,cyclosporine), an angiogenesis inhibitor, an antibiotic, and a standardof care regimen for the treatment of dry eye disease.

A. Standard of Care for Regimen for Treatment of Dry Eye Disease

In one embodiment, methods described herein optionally compriseadministering a standard of care therapeutic to the subject. In someembodiments, the standard of care therapeutic and theanti-lymphangiogenic agent are co-administered in a single composition.In other embodiments, the standard of care therapeutic is administeredas a separate composition from the anti-lymphangiogenic agent.

In the context of methods of the invention, “standard of care” refers toa treatment that is generally accepted by clinicians for a certain typeof patient diagnosed with a type of illness. For dry eye disease, forexample, an aspect of the invention is to improve standard of caretherapy with co-therapy with anti-lymphangiogenic agents describedherein that inhibit lymphangiogenesis. Exemplary standard of careregimens for dry eye disease include, but are not limited to, eyelidhygiene, topical antibiotics (including, but not limited to erythromycinor bacitracin ointments), oral tetracyclines (tetracycline, doxycycline,or minocycline), anti-inflammatory compounds (including, but not limitedto, cyclosporine) and corticosteroids.

B. VEGFR-2 Inhibitor Product(s)

In one embodiment, methods described herein optionally compriseadministering a VEGFR-2 inhibitor product to the subject. In someembodiments, the VEGFR-2 inhibitor product and the anti-lymphangiogensisagent are co-administered in a single composition. In other embodiments,the VEGFR-2 inhibitor product is administered as a separate compositionfrom the anti-lymphangiogenesis agent. cDNA and amino acid sequences ofhuman VEGFR-2 are set forth in SEQ ID NOs: 50 and 51, respectively. The“VEGFR-2 inhibitor product” can be any molecule that acts withspecificity to reduce VEGF-C/VEGFR-2, VEGF-D/VEGFR-2 or VEGF/VEGFR-2interactions, e.g., by blocking VEGF-C or VEGF-D binding to VEGFR-2, byblocking VEGF binding to VEGFR-2 or by reducing expression of VEGFR-2.In one embodiment, the VEGFR-2 inhibitor inhibits VEGF-C and VEGF-Dbinding to VEGFR-2. In another embodiment, the VEGFR-2 inhibitorinhibits binding of VEGF to VEGFR-2. The VEGFR-2 inhibitor can be apolypeptide comprising a soluble VEGFR-2 extracellular domain fragment(amino acids 20-764 of SEQ ID NO: 51) that binds VEGF or VEGF-C orVEGF-D; VEGFR-2 anti-sense polynucleotides or short-interfering RNA(siRNA); anti-VEGFR-2 antibodies; a VEGFR-2 inhibitor polypeptidecomprising an antigen-binding fragment of an anti-VEGFR-2 antibody thatinhibits binding between VEGFR-2 and VEGF or VEGF-C or VEGF-D; anaptamer that inhibits binding between VEGFR-2 and VEGF; an aptamer thatinhibits binding between VEGFR-2 and VEGF-C; an aptamer that inhibitsbinding between VEGFR-2 and VEGF-D; or a fusion protein comprising thesoluble VEGFR-2 polypeptide fragment fused to an immunoglobulin constantregion fragment (Fc). In some embodiments, a VEGFR-2 polypeptidefragment is fused to alkaline phosphatase (AP). Methods for making Fc orAP fusion constructs are found in WO 02/060950, the disclosure of whichis incorporated herein by reference in its entirety.

A number of VEGFR-2 antibodies have been described, see for example,U.S. Pat. No. 6,334,339 and U.S. Patent Publication Nos. 2002/0064528,2005/0214860, and 2005/0234225 (all of which are incorporated herein byreference in their entireties). Antibodies are useful for modulatingVEGFR-2/VEGF interactions due to the ability to easily generateantibodies with relative specificity, and due to the continuedimprovements in technologies for adopting antibodies to human therapy.Thus, the invention contemplates use of antibodies (e.g., monoclonal andpolyclonal antibodies, single chain antibodies, chimeric antibodies,bifunctional/bispecific antibodies, humanized antibodies, humanantibodies, and complementary determining region (CDR)-graftedantibodies, including compounds which include CDR sequences whichspecifically recognize a polypeptide of the invention) specific forVEGFR-2. Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86 95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779 783(1992); Lonberg et al., Nature 368 856 859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13:65-93 (1995).

VEGFR-2 antibody fragments, including Fab, Fab′, F(ab′)2, Fv, scFv, arealso contemplated. The term “specific for,” when used to describeantibodies of the invention, indicates that the variable regions of theantibodies of the invention recognize and bind the polypeptide ofinterest exclusively (i.e., able to distinguish the polypeptides ofinterest from other known polypeptides of the same family, by virtue ofmeasurable differences in binding affinity, despite the possibleexistence of localized sequence identity, homology, or similaritybetween family members). It will be understood that specific antibodiesmay also interact with other proteins (for example, S. aureus protein Aor other antibodies in ELISA techniques) through interactions withsequences outside the variable region of the antibodies, and inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of an antibody of the invention are wellknown and routinely practiced in the art. For a comprehensive discussionof such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual;Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter6. Antibodies of the invention can be produced using any method wellknown and routinely practiced in the art.

In another embodiment, methods described herein optionally compriseadministering an anti-sense VEGFR-2 nucleic acid molecule to thesubject. Anti-sense VEGFR-2 nucleic acid molecules are usefultherapeutically to inhibit the translation of VEGFR-2-encoding mRNAswhere the therapeutic objective involves a desire to eliminate thepresence of VEGFR-2 or to downregulate its levels. VEGFR-2 anti-senseRNA, for example, could be useful as a VEGFR-2 antagonizing agent in thetreatment of diseases in which VEGFR-2 is involved as a causative agent,e.g, inflammatory diseases.

An antisense nucleic acid comprises a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding a protein (e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence). (See, e.g., the VEGFR-3 cDNAsequence of SEQ ID NO: 9). Methods for designing and optimizingantisense nucleotides are described in Lima et al., (J Biol Chem;272:626-38. 1997) and Kurreck et al., (Nucleic Acids Res.; 30:1911-8.2002). In specific aspects, antisense nucleic acid molecules areprovided that comprise a sequence complementary to at least about 10,25, 50, 100, 250 or 500 nucleotides or an entire VEGFR-2 coding strand,or to only a portion thereof. Nucleic acid molecules encoding fragments,homologs, derivatives and analogs of a VEGFR-2 or antisense nucleicacids complementary to a VEGFR-2 nucleic acid sequence are alsocontemplated.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encoding aVEGFR-2 protein. The term “coding region” refers to the region of thenucleotide sequence comprising codons which are translated into aminoacid residues. In another embodiment, the antisense nucleic acidmolecule is antisense to a “conceding region” of the coding strand of anucleotide sequence encoding the VEGFR-2. The term “conceding region”refers to 5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

Antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick or Hoogsteen base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof VEGFR-2 mRNA, but more preferably is an oligonucleotide that isantisense to only a portion of the coding or noncoding region of VEGFR-2mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15,20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis or enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids (e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used).

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation.

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding VEGFR-2 tothereby inhibit expression of the protein (e.g., by inhibitingtranscription and/or translation). The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule that binds toDNA duplexes, through specific interactions in the major groove of thedouble helix.

In still another embodiment, VEGFR-2 RNA can be used for induction ofRNA interference (RNAi), using double stranded (dsRNA) (Fire et al.,Nature 391: 806-811. 1998) or short-interfering RNA (siRNA) sequences(Yu et al., Proc Natl Acad Sci USA. 99:6047-52. 2002). “RNAi” is theprocess by which dsRNA induces homology-dependent degradation ofcomplimentary mRNA. In one embodiment, a nucleic acid molecule of theinvention is hybridized by complementary base pairing with a “sense”ribonucleic acid of the invention to form the double stranded RNA. ThedsRNA antisense and sense nucleic acid molecules are provided thatcorrespond to at least about 20, 25, 50, 100, 250 or 500 nucleotides oran entire VEGFR-2 coding strand, or to only a portion thereof. In analternative embodiment, the siRNAs are 30 nucleotides or less in length,and more preferably 21- to 23-nucleotides, with characteristic 2- to3-nucleotide 3′-overhanging ends, which are generated by ribonucleaseIII cleavage from longer dsRNAs. See e.g. Tuschl T. (Nat Biotechnol.20:446-48. 2002). Preparation and use of RNAi compounds is described inU.S. Patent Publication No. 2004/0023390, the disclosure of which isincorporated herein by reference in its entirety.

Intracellular transcription of small RNA molecules can be achieved bycloning the siRNA templates into RNA polymerase III (Pol III)transcription units, which normally encode the small nuclear RNA (snRNA)U6 or the human RNAse P RNA H1. Two approaches can be used to expresssiRNAs: in one embodiment, sense and antisense strands constituting thesiRNA duplex are transcribed by individual promoters (Lee, et al. Nat.Biotechnol. 20, 500-505. 2002); in an alternative embodiment, siRNAs areexpressed as stem-loop hairpin RNA structures that give rise to siRNAsafter intracellular processing (Brummelkamp et al. Science 296:550-553.2002) (incorporated herein by reference).

The dsRNA/siRNA is most commonly administered by annealing sense andantisense RNA strands in vitro before delivery to the organism. In analternate embodiment, RNAi may be carried out by administering sense andantisense nucleic acids of the invention in the same solution withoutannealing prior to administration, and may even be performed byadministering the nucleic acids in separate vehicles within a very closetimeframe. Nucleic acid molecules encoding fragments, homologs,derivatives and analogs of a VEGFR-2 or antisense nucleic acidscomplementary to a mVEGFR-2 nucleic acid sequence are also contemplated.

Aptamers are another nucleic acid based method for interfering with theinteraction of VEGFR-2 or VEGFR-3 and their respective ligands VEGF-Aand/or VEGF-C and/or VEGF-D. Aptamers are DNA or RNA molecules that havebeen selected from random pools based on their ability to bind othermolecules. Aptamers have been selected which bind nucleic acid,proteins, small organic compounds, and even entire organisms. Methodsand compositions for identifying and making aptamers are known to thoseof skill in the art and are described e.g., in U.S. Pat. No. 5,840,867and U.S. Pat. No. 5,582,981 each incorporated herein by reference.

Recent advances in the field of combinatorial sciences have identifiedshort polymer sequences with high affinity and specificity to a giventarget. For example, SELEX technology has been used to identify DNA andRNA aptamers with binding properties that rival mammalian antibodies,the field of immunology has generated and isolated antibodies orantibody fragments which bind to a myriad of compounds and phage displayhas been utilized to discover new peptide sequences with very favorablebinding properties. Based on the success of these molecular evolutiontechniques, it is certain that molecules can be created which bind toany target molecule. A loop structure is often involved with providingthe desired binding attributes as in the case of: aptamers which oftenutilize hairpin loops created from short regions without complimentarybase pairing, naturally derived antibodies that utilize combinatorialarrangement of looped hyper-variable regions and new phage displaylibraries utilizing cyclic peptides that have shown improved resultswhen compared to linear peptide phage display results. Thus, sufficientevidence has been generated to suggest that high affinity ligands can becreated and identified by combinatorial molecular evolution techniques.For the present invention, molecular evolution techniques can be used toisolate binding constructs specific for ligands described herein. Formore on aptamers, See generally, Gold, L., Singer, B., He, Y. Y., Brody.E., “Aptamers As Therapeutic And Diagnostic Agents,” J. Biotechnol.74:5-13 (2000). Relevant techniques for generating aptamers may be foundin U.S. Pat. No. 6,699,843, which is incorporated by reference in itsentirety.

In some embodiments, the aptamer may be generated by preparing a libraryof nucleic acids; contacting the library of nucleic acids with a growthfactor, wherein nucleic acids having greater binding affinity for thegrowth factor (relative to other library nucleic acids) are selected andamplified to yield a mixture of nucleic acids enriched for nucleic acidswith relatively higher affinity and specificity for binding to thegrowth factor. The processes may be repeated, and the selected nucleicacids mutated and re-screened, whereby a growth factor aptamer is beidentified.

In yet another variation, the VEGFR-2 inhibitor product comprises asoluble extracellular domain fragment of VEGFR-1 that binds VEGF andinhibits VEGF binding to VEGFR-2. cDNA and amino acid sequences ofVEGFR-1 are set forth in SEQ ID NOs: 18 and 19. Exemplary extracellulardomain fragments of VEGFR-1 are described in U.S. Patent Publication No.2006/0030000 and International Patent Publication No. WO 2005/087808,the disclosures of which are incorporated herein by reference in theirentireties.

C. Anti-inflammatory Agents

In another embodiment, the methods described herein optionally compriseadministering one or more anti-inflammatory agents to the subject. Insome embodiments, the anti-inflammatory agent and theanti-lymphangiogenic agent are co-administered in a single composition.In other embodiments, the anti-inflammatory agent is administered as aseparate composition from the anti-lymphangiogenic agent. Combinationsinvolving an anti-lymphangiogenic agent, a VEGFR-2 inhibitor, and ananti-inflammatory agent are specifically contemplated. As used herein,the term “anti-inflammatory agent” refers generally to any agent thatreduces inflammation or swelling in a subject. A number of exemplaryanti-inflammatory agents are recited herein, but it will be appreciatedthat there may be additional suitable anti-inflammatory agents notspecifically recited herein, but which are encompassed by the presentinvention.

In one variation, the anti-inflammatory agent is a non-steroidalanti-inflammatory drug (NSAID). Exemplary NSAIDs include, but are notlimited to: aspirin, Sulfasalazine™, Asacol™, Dipendtum™, Pentasa™,Anaprox™, Anaprox DS™ (naproxen sodium); Ansaid™ (flurbiprofen);Arthrotec™ (diclofenac sodium+ misoprostil); Cataflam™/Voltaren™(diclofenac potassium); Clinoril™ (sulindac); Daypro™ (oxaprozin);Disalcid™ (salsalate); Dolobid™ (diflunisal); EC Naprosyn™ (naproxensodium); Feldene™ (piroxicam); Indocin™, Indocin SR™ (indomethacin);Lodine™, Lodine XL™ (etodolac); Motrin™ (ibuprofen); Naprelan™(naproxen); Naprosyn™ (naproxen); Orudis™, (ketoprofen); Oruvail™(ketoprofen); Relafen™ (nabumetone); Tolectin™, (tolmetin sodium);Trilisate (choline magnesium trisalicylate); Cox-1 inhibitors; Cox-2Inhibitors such as Vioxx™ (rofecoxib); Arcoxia™ (etoricoxib), Celebrex™(celecoxib); Mobi™ (meloxicam); Bextra™ (valdecoxib), Dynastat™paracoxib sodium; Prexige™ (lumiracoxib), and nambumetone. Additionalsuitable NSAIDs, include, but are not limited to, the following:5-aminosalicyclic acid (5-ASA, mesalamine, lesalazine),ε-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, anitrazafen, antrafenine, bendazac, bendazac lysinate,benzydamine, beprozin, broperamole, bucolome, bufezolac, ciproquazone,cloximate, dazidamine, deboxamet, detomidine, difenpiramide,difenpyramide, difisalamine, ditazol, emorfazone, fanetizole mesylate,fenflumizole, floctafenine, flumizole, flunixin, fluproquazone,fopirtoline, fosfosal, guaimesal, guaiazolene, isonixirn, lefetamineHCl, leflunomide, lofemizole, lotifazole, lysin clonixinate,meseclazone, nabumetone, nictindole, nimesulide, orgotein, orpanoxin,oxaceprolm, oxapadol, paranyline, perisoxal, perisoxal citrate,pifoxime, piproxen, pirazolac, pirfenidone, proquazone, proxazole,thielavin B, tiflamizole, timegadine, tolectin, tolpadol, tryptamid andthose designated by company code number such as 480156S, AA861, AD1590,AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CN100,EB382, EL508, F1044, FK-506, GV3658, ITF182, KCNTEI6090, KME4, LA2851,MR714, MR897, MY309, ONO3144, PR823, PV102, PV108, R830, RS2131, SCR152,SH440, SIR133, SPAS510, SQ27239, ST281, SY6001, TA60, TAI-901(4-benzoyl-1-indancarboxylic acid), TVX2706, U60257, UR2301 and WY41770.

In another variation, the anti-inflammatory agent can be a compound thatinhibits the interaction of inflammatory cytokines with their receptors.Examples of cytokine inhibitors useful in combination with the specificbinding agents of the invention include, for example, antagonists (suchas antibodies) of TGF-α (e.g., Remicade), as well as antagonists (suchas antibodies) directed against interleukins involved in inflammation.Such interleukins are described herein and preferably include, but arenot limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-12,IL-13, IL-17, and IL-18. See Feghali, et al., Frontiers in Biosci.,2:12-26 (1997).

In another variation, the anti-inflammatory agent is a corticosteroid.Exemplary corticosteroids include, but are not limited to, difloroasonediacetate, clobetasol propionate, halobetasol propionate, betamethasone,betamethasone dipropionate, budesonide, cortisone, dexamethasone,fluocinonide, halcinonide desoximethasone, triamcinolone, fluticasonepropionate, fluocinolone acetonide, flurandrenolide, mometasone furoate,betamethosone, fluticasone propionate, fluocinolone acetonide,aclometasome dipropionate, methylprednisolone, prednisolone, prednisone,triamicinolone, desonide and hydrocortisone.

In another variation, the anti-inflammatory agent is cyclosporine.

D. Antibiotics

In another embodiment, the methods described herein optionally furthercomprise administering an antibiotic to the subject. In someembodiments, the antibiotic and the anti-lymphangiogenic agent areco-administered in a single composition. In other embodiments, theantibiotic is administered as a separate composition from theanti-lymphangiogenic agent. Exemplary antibiotics include, but are notlimited to, tetracycline, aminoglycosides, penicillins, cephalosporins,sulfonamide drugs, chloramphenicol sodium succinate, erythromycin,vancomycin, lincomycin, clindamycin, nystatin, amphotericin B,amantidine, idoxuridine, p-amino salicyclic acid, isoniazid, rifampin,antinomycin D, mithramycin, daunomycin, adriamycin, bleomycin,vinblastine, vincristine, procarbazine, and imidazole carboxamide.

D. Tyrosine Kinase Inhibitors

In another embodiment, the methods described herein optionally furthercomprise administering a tyrosine kinase inhibitor that inhibits VEGFR-2and/or VEGFR-3 activity. It is contemplated that VEGFR-3 tyrosine kinaseinhibitors would be useful in combination therapy embodiments where theanti-lymphangiogenic agent is not a VEGFR-3 tyrosine kinase inhibitor.

Exemplary tyrosine kinase inhibitors for use in the methods describedherein include, but are not limited to, AEE788 (TKI, VEGFR-2, EGFR:Novartis); ZD6474 (TKI, VEGFR-1, -2,-3, EGFR: Zactima: AstraZeneca);AZD2171 (TKI, VEGFR-1, -2: AstraZeneca); SU 11248 (TKI, VEGFR-1, -2,PDGFR: Sunitinib: Pfizer); AG13925 (TKI, VEGFR-1, -2: Pfizer); AG013736(TKI, VEGFR-1, -2: Pfizer); CEP-7055 (TKI, VEGFR-1, -2,-3: Cephalon);CP-547,632 (TKI, VEGFR-1, -2: Pfizer); GW7S6024 (TKL VEGFR-1, -2, -3:GlaxoSmithKline); GW786034 (TKI, VEGFR-1, -2, -3: GlaxoSmithKline);sorafenib (TKI, Bay 43-9006, VEGFR-1, -2, PDGFR: Bayer/Onyx); SU4312(TKI, VEGFR-2, PDGFR: Pfizer); AMG706 (TKI, VEGFR-1, -2, -3: Amgen);XL647 (TKI, EGFR, HER2, VEGFR, ErbB4: Exelixis); XL999 (TK1, FGFR,VEGFR, PDGFR, F11-3: Exelixis); PKC412 (TKI, KIT, PDGFR, PKC, FLT3,VEGFR-2: Novartis); AEE788 (TKI, EGFR, VEGFR2, VEGFR-1: Novartis):OSI-030 (TKI, c-kil, VEGFR: OSI Pharmaceuticals); OS 1-817 (TKI c-kit,VEGFR: OSI Pharmaceuticals); DMPQ (TKI, ERGF, PDGFR, ErbB2. p56. pkA,pkC); MLN518 (TKI, F1t3, PDGFR, c-KIT (T53518: MillenniumPharmaceuticals); lestaurinib (TKI, FLT3, CEP-701, Cephalon); ZD 1839(TKI, EGFR: gefitinib, Iressa: AstraZcneca); OSI-774 (TKI, EGFR:Erlotininb: Tarceva: OSI Pharmaceuticals); lapatinib (TKI, ErbB-2, EGFR,and GD-2016: Tykerb: GlaxoSmithKline).

In some embodiments, the methods described herein further compriseadministering a tyrosine kinase inhibitor that inhibits angiogenesis tothe subject. Exemplary anti-angiogeneic tyrosine kinase inhibits andtheir targets are provided below in Table 2.

TABLE 2 Antiangiogenic tyrosine kinase receptor inhibitors and theirtargets Agent VEGFR-1 VEGFR-2 VEGFR-3 PDGFR EGFR Other targetsVandetanib   RET Sunitinib     KIT, FLT3, RET Axitinib   Sorafenib     KIT, RAF, FLT3 Vatalanib     KIT Cediranib    KIT Motesanib     KIT, RET Pazopanib     KIT BIBF 1120   FGFRAbbreviations: FGFR, fibroblast-like growth factor receptor; FLT3,FMS-like tyrosine kinase 3; KIT, stem cell factor receptor; RET, glialcell line-derived neurotrophic factor receptor; VEGFR, vascularendothelial growth factor receptor.

E. Administration of the Combination Therapy

Combination therapy with one or more of the additional agents describedherein may be achieved by administering to a subject a singlecomposition or pharmacological formulation that includes theanti-lymphangiogenic agent and the one or more additional agents, or byadministering to the subject two (or more) distinct compositions orformulations, at the same time, wherein one composition includes ananti-lymphangiogenic agent and the other includes a second agent.

Alternatively, the combination therapy employing an anti-lymphangiogenicagent described herein may precede or follow the second agent treatmentby intervals ranging from minutes to weeks. In embodiments where thesecond agent and the anti-lymphangiogenic agent are administeredseparately, one would generally ensure that a significant period of timedid not expire between the times of each delivery, such that the agentand the anti-lymphangiogenic agent would still be able to exert anadvantageously combined effect. In such instances, it is contemplatedthat one would administer both modalities within about 12-24 hours ofeach other and, more preferably, within about 6-12 hours of each other,with a delay time of only about 12 hours being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations. Repeated treatments with one or both agents isspecifically contemplated.

Formulations and Pharmaceutically Acceptable Carriers

Some exemplary ophthalmic viscosity enhancers that can be used in thepresent formulation include: carboxymethyl cellulose sodium;methylcellulose; hydroxypropyl cellulose; hydroxypropylmethyl cellulose;hydroxyethyl cellulose; polyethylene glycol 300; polyethylene glycol400; polyvinyl alcohol; and providone.

Some natural products, such as veegum, alginates, xanthan gum, gelatin,acacia and tragacanth, may also be used to increase the viscosity ofophthalmic solutions.

A tonicity is important because hypotonic eye drops cause an edema ofthe cornea, and hypertonic eye drops cause deformation of the cornea.The ideal tonicity is approximately 300 mOsM. The tonicity can beachieved by methods described in Remington: The Science and Practice ofPharmacy, known to those versed in the art.

Suitable ophthalmic carriers are known to those skilled in the art andall such conventional carriers may be employed in the present invention.Exemplary compounds incorporated to facilitate and expedite transdermaldelivery of topical compositions into ocular or adnexal tissues include,but are not limited to, alcohol (ethanol, propanol, and nonanol), fattyalcohol (lauryl alcohol), fatty acid (valeric acid, caproic acid andcapric acid), fatty acid ester (isopropyl myristate and isopropyln-hexanoate), alkyl ester (ethyl acetate and butyl acetate), polyol(propylene glycol, propanedione and hexanetriol), sulfoxide(dimethylsulfoxide and decylmethylsulfoxide), amide (urea,dimethylacetamide and pyrrolidone derivatives), surfactant (sodiumlauryl sulfate, cetyltrimethylannmonium bromide, polaxamers, spans,tweens, bile salts and lecithin), terpene (d-limonene, alphaterpeneol,1,8-cineole and menthone), and alkanone (N-heptane and N-nonane).Moreover, topically-administered compositions comprise surface adhesionmolecule modulating agents including, but not limited to, a cadherinantagonist, a selectin antagonist, and an integrin antagonist. Thus, aparticular carrier may take the form of a sterile, ophthalmic ointment,cream, gel, solution, or dispersion. Also including as suitableophthalmic carriers are slow release polymers, e.g., “Ocusert” polymers,“Hydron” polymers, etc.

Stabilizers may also be used such as, for example, chelating agents,e.g., EDTA. Antioxidants may also be used, e.g., sodium bisulfite,sodium thiosulfite, 8-hydroxy quinoline or ascorbic acid. Sterilitytypically will be maintained by conventional ophthalmic preservatives,e.g., chiorbutanol, benzalkonium chloride, cetylpyridium chloride,phenyl mercuric salts, thimerosal, etc., for aqueous formulations, andused in amounts which are nontoxic and which generally vary from about0.001 to about 0.1% by weight of the aqueous solution. Conventionalpreservatives for ointments include methyl and propyl parabens. Typicalointment bases include white petrolatum and mineral oil or liquidpetrolatum. However, preserved aqueous carriers are preferred. Solutionsmay be manually delivered to the eye in suitable dosage form, e.g., eyedrops, or delivered by suitable microdrop or spray apparatus typicallyaffording a metered dose of medicament. Examples of suitable ophthalmiccarriers include sterile, substantially isotonic, aqueous solutionscontaining minor amounts, i.e., less than about 5% by weighthydroxypropylmethylcellulose, polyvinyl alcohol, carboxymethylcellulose,hydroxyethylcelullose, glycerine and EDTA. The solutions are preferablymaintained at substantially neutral pH and isotonic with appropriateamounts of conventional buffers, e.g., phosphate, borate, acetate, tris.

In some embodiments, penetration enhancers are added to theophthalmologic carrier.

Routes of Administration

The composition containing the anti-lymphangiogenic agent can beadministered to a patient by a variety of means depending, in part, onthe type of agent to be administered and the history, risk factors andsymptoms of the patient. Routes of administration suitable for themethods of the invention include both systemic and local administration.Thus, in one embodiment, a method of treating dry eye disease ispracticed by systemic administration of a pharmaceutical compositioncomprising the anti-lymphangiogenic agent. In another embodiment, amethod of the invention is practiced by local administration of apharmaceutical composition comprising the anti-lymphangiogenic agent. Infurther embodiments, a pharmaceutical composition comprising theanti-lymphangiogenic agent is administered topically, or by localinjection, or is released from an intraocular or periocular implant.

As used herein, the term “systemic administration” means a mode ofadministration resulting in delivery of a pharmaceutical composition toessentially the whole body of the patient. Exemplary modes of systemicadministration include, without limitation, intravenous injection andoral administration. The term “local administration,” as used herein,means a mode of administration resulting in significantly morepharmaceutical composition being delivered to and about the eyes than toregions distal from the eyes.

Systemic and local routes of administration useful in the methods of theinvention encompass, without limitation, oral gavage; intravenousinjection; intraperitoneal injection; intramuscular injection;subcutaneous injection; transdermal diffusion and electrophoresis;topical eye drops and ointments; periocular and intraocular injectionincluding subconjunctival injection; extended release delivery devicesincluding locally implanted extended release devices; and intraocularand periocular implants including bioerodible and reservoir-basedimplants.

In some embodiments, an ophthalmic composition containing ananti-lymphangiogenic agent is administered topically to the eye. Theophthalmic composition can be for example, an ophthalmic solution(ocular drops). In other embodiments, the ophthalmic compositioncontaining the anti-lymphangiogenic agent is injected directly into theeye. In a further embodiment, the ophthalmic composition containing theanti-lymphangiogenic agent is released from an intraocular or periocularimplant such as a bioerodible or reservoir-based implant.

In some embodiments, the ophthalmic composition comprising ananti-lymphangiogenic agent is administered locally in an extendedrelease formulation. For example, an ophthalmic composition containingan anti-lymphangiogenic agent can be administered via an intraocular orperiocular implant, which can be, for example, bioerodible orreservoir-based. As used herein, the term “implant” refers to anymaterial that does not significantly migrate from the insertion sitefollowing implantation. An implant can be biodegradable,non-biodegradable, or composed of both biodegradable andnon-biodegradable materials; a non-biodegradable implant can include, ifdesired, a refillable reservoir. Implants useful in the methods of theinvention include, for example, patches, particles, sheets, plaques,microcapsules and the like, and can be of any shape and size compatiblewith the selected site of insertion, which can be, without limitation,the posterior chamber, anterior chamber, suprachoroid or subconjunctiva.It is understood that an implant useful in the invention generallyreleases the implanted pharmaceutical composition at an effective dosageto the eye of the patient over an extended period of time. A variety ofocular implants and extended release formulations suitable for ocularrelease are well known in the art, as described, for example, in U.S.Pat. Nos. 5,869,079 and 5,443,505.

In instances where the anti-lymphangiogenic agent is a nucleic acidmolecule, administration of a pharmaceutical composition containing thenucleic acid molecule can be carried out using one of numerous methodswell known in the art of gene therapy. Such methods include, but are notlimited to, lentiviral transformation, adenoviral transformation,cytomegaloviral transformation, microinjection and electroporation.

In some embodiments, the anti-lymphangiogenic agents are administered tothe subject in a liquid or gel suspension in the form of drops, spray orgel. In yet another embodiment, the anti-lymphangiogenic agents areinjected directly into the lacrimal tissues or onto the eye surface.

In other embodiments, the anti-lymphangiogenic agents are applied to theeye via liposomes. In still other embodiments, the anti-lymphangiogenicagents are infused into the tear film via a pump-catheter system. Instill another embodiment, the anti-lymphangiogenic agents are containedwithin a continuous or selective-release device, for example, membranessuch as, but not limited to, those employed in the Ocusert™ System (AlzaCorp., Palo Alto, Calif.). As an additional embodiment, theanti-lymphangiogenic agents are contained within, carried by, orattached to contact lenses which are placed on the eye. In yet anotherembodiment, the anti-lymphangiogenic agents are contained within a swabor sponge which can be applied to the ocular surface. Another embodimentof the present invention involves the active compound contained within aliquid spray which can be applied to the ocular surface.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EXAMPLES Materials and Methods Experimental Dry Eye Murine Model

Eight to ten week-old female C57BLI6 mice (Charles River Laboratory,Wilmington, Mass.) were used in accordance with the standards in theARVO Statement for the Use of Animals in Ophthalmic and Vision Research.The research protocol was approved by the Schepens Eye ResearchInstitute Animal Care and Use Committee. Dry eye was induced in murineeyes using a Controlled Environment Chamber (CEC) which exposes the miceto high-flow desiccated air. To achieve maximum ocular surface dryness,the conditions in CEC were supplemented with topical application of 1%atropine sulfate (Falcon Pharma, Fort Worth, Tex.) twice for the first48 hours and subcutaneous injections of 0.1 ml of 5 mg/ml of scopolaminehydrobromide (Sigma-Aldrich, St. Louis, Mo.) three times a day, for theentire duration of the experiment.

RNA Isolation and Molecular Analysis Using Real Time Polymerase ChainReaction

Five mice (10 eyes) were included in each group. Two corneas were pooledtogether to equal as one sample and stored at −80′C in Trizol(Invitrogen, Carlsbad, Calif.; catalog No. 15596026) until future use.Total RNA was isolated from these corneas using the RNeasy microkit(Qiagen, Valencia, Calif.; catalog No. 74004). Equal amounts of RNA wereused to synthesize cDNA using SuperScript™ III Reverse Trancriptase(Invitrogen, Carlsbad, Calif.; catalog No. 18080) according to themanufacturer's recommendations. Real-Time PCR was performed usingFAM-MGB dye labeled predesigned primers (Applied Biosystem, Foster City,Calif.) for GAPDH (assay ID.Mm999999 15_g1), VEGF-A (Mm00437304_m1),VEGF-C(Mrn00437313_m1), VEGF-D (Mm00438965_m1), VEGFR-2 (Mm00440099_m1),VEGFR-3 (Mm00433337_m1). 2.5 μl of cDNA was loaded in each well andassays were performed in duplicate. The GAPDH gene was used as theendogenous reference for each reaction. The results were normalized bythe cycle threshold (CT) of GAPDH and the relative mRNA level in thenormal mice was used as the normalized control.

Immunohistochemistry

The following primary antibodies were used for immunohistochemicalstaining: rat anti-mouse CD11b-FITC for monocytes/macrophages (BDPharmingen, San Diego, Calif., 1:100), goat anti-mouse CD31 FITC aspan-endothelial marker (Santa Cruz Biotechnology, Santa Cruz, Calif., 1:100) and purified rabbit anti-mouse LYVE-1 as iymphatic endothelialmarker (Abeam, MA, USA, 1:400). Respective isotypes were used asnegative controls. Rhodamine conjugated goat anti-rabbit (BD Pharmingen,San Diego, Calif., 1:100) was the secondary antibody used.

Freshly excised corneas were washed in PBS, fixed in acetone for 15minutes and then double stained with CD31 and LYVE-1 as describedpreviously. To analyze infiltration of CD11b⁺/LYVE-1 cells, corneas fromthree mice from each group were taken and cells were counted in 5-6areas in the periphery (0.5 μm area from the limbus) of each cornea in amasked fashion, using epifluorescence microscope (model E800; Nikon,Melville, N.Y.) at 40× magnification. The mean number of cells wasobtained by averaging the total number of cells in all the areas studiedand the result was expressed as the number of positive cells per mm².

Morphometry of Lymphangiogenesis in the Cornea

Morphology of lymphatics was analyzed using an automated image analysisprogram written with Matlab (The Mathworks, Inc., Natick, Mass.).Lymphatics were isolated from digitized images with this program usingstandard computer vision techniques for image segmentation, includingbackground isolation and subtraction, edge detection, and k-meansclustering. This segmentation process generated binary images in whichlymphatic vessels are represented by is and all other image content isrepresented by Os. The resultant isolated lymphatic vessels wereanalyzed morphologically using two metrices, Lymphatic Area (LA) andLymphatic Caliber (LC). LA represents the total surface area of thelymphatic vessels when projected into the plane of the image. LC is asummary measure of the diameters of the lymphatic vessels present. LCwas measured using a computational technique that generates the largestdiameter circle centered at each pixel inside a lymphatic vessel. Themean value across all pixels within lymphatic vessels was taken as anestimate of the mean LC for a given image.

Flow Cytometry

Draining LNs from DED (day 10) and normal mice were collected. Singlecell suspension of LN cells was stained with the anti-CDI 1b-FITC andanti-lab (MHC-II)-PE. Stained LN cells were then analyzed on an EPICS XLflow cytometer (Beckman Coulter). All the antibodies with their matchedisotype controls were purchased from eBioscience.

Studies involving inhibition of corneal neo-lymphangiogenesis using ananti-VEGF-C antibody (Example 5 onwards)

Anti-VEGF-C antibodies (VGX-100; Vegenics Limited, Australia) wereadministered intraperitonealy daily from day 1 to day 10 to DED mice.Mice were assessed clinically using corneal fluorescent staining.Tissues from cornea, conjunctiva and draining lymph nodes were examinedfor cellular and molecular pathological changes. In vivo blockade ofVEGF-C suppresses corneallymphangiogenesis and ameliorates clinicalsigns of DED.

Statistical Analysis

A two-tailed Student's t-test was performed and P-values less than 0.05were deemed statistically significant. Results are presented as themean±SEM of at least three experiments.

Example 1 Demonstration and Quantification of Lymphatics in Dry EyeCorneas

To determine whether DED induces growth of lymphatics into the cornea,and whether lymphatic growth is paralleled by growth of blood vessels,corneal whole mounts were double stained for CD31 (pan-endothelialmarker) and LYVE-1 (lymphatic vascular endothelial marker) at days 0, 6,10 and 14 and quantified for lymphangiogenesis. Blood vessels wereidentified as CD31^(hi)/LyvE-1⁻ and lymph vessels were identified asCD31^(lo)/LYVE-1^(hi). A significant increase in lymphatic area LA isseen in DED mice (FIG. 1 b). Morphometric analysis revealed small budsof lymphatic vessels arising from the limbal vascular arcade at an earlytime point (day 6), which increased in caliber (LC) and area (LA), andadvanced towards the center of the cornea with DED progression (FIGS. 1and 2). A significant increase in LA (FIG. 3a ) was seen as early as day6 (P<0.01) which continued until day 14 (P<0.0001). However, LC (FIG. 3b) was significantly increased from the normal only by day 14 (P<0.02).Remarkably, these lymphatics were not accompanied by growth of bloodvessels at any given time point.

Example 2 Expression Levels of Different VEGF's and VEGFR's in Dry EyeCorneas

The development of lymphatic vessels is regulated by factors common toboth hemangiogenesis and lymphangiogenesis. VEGF-C and VEGF-D are theclassic lymphangiogenic factors and act by binding to their receptorsVEGFR-2 and VEGFR-3, which are expressed on lymphatic endothelial cells.To determine the molecular mechanisms of lymphangiogenesis in DED,expression of different vascular endothelial growth factors and theirreceptors were quantified at indicated time points in the cornea usingreal time PCR. Amongst the VEGF species (FIG. 4), lymphangiogenicspecific VEGF-D was not only the earliest to increase at day 6 (−2folds; P<0.03) but also showed the maximum increase in expression at day14 (−3 folds; P<0.03). Significant increased transcript expression ofVEGF-A and VEGF-C was seen only by day 14 (P<0.03 for both). Similarlylevels of lymphangiogenic specific VEGFR-3 were first to show asignificant increase at day 6 (−4 folds; P<0.01) and continued to riseuntil day 14 (−8 folds; P<0.01). Though an overall trend towardincreased expression was noticed with VEGFR-2 (primarily specific forblood vessel growth), significant increase (P<0.05) was appreciated onlyby day 14 (FIG. 5).

Example 3 Enumeration of CD11b/LYVE-1 Positive Cells in Dry Eye Corneas

The normal cornea has a resident population of bone marrow-derivedCD11b⁺ monocyticmacrophage-lineage cells and the development of DEDincreases the number of CD11b⁺ cells in the cornea. The role ofmacrophages in inflammatory lymphangiogenesis is well established. TheseCD11b⁺ macrophages may also express various lymphatic endothelialmarkers, such as LYVE-1. To see what proportion of these CD11b⁺ cellshad lymphangiogenic potential, whole mount corneal tissues were doublestained with CD11b and LYVE-1 at day 14. There was a significantincrease in the number of both CD11b+(P<0.02) and CD11b⁺/LYVE-1⁺(P<0.0001) cells in dry eye as compared to normal corneas (FIG. 6). InDED, about 25% of the CD11b⁺ cells were positive for LYVE-1 where asonly 4% of the CD11b⁺ cells were positive for LYVE-1 in the normalcorneas.

Example 4 Role of APC Homing

It was next investigated whether corneal lymphangiogenesis in DED isassociated with the increased homing of APC in the draining LN. Usingflow cytometry, the frequencies of mature APC (MHC-II+CD11b+) in thedraining LN of normal and DED mice were analysed (FIG. 7). Data showed asignificant increase in the frequency of MHC-II+CD11b⁺ APC in the LNcells of DED mice compared to those in the LN of normal mice (Range:14.9-19.5% vs. 10-13.5%, p<0.05).

Example 5 Effect of In Vivo Blockade of Pro-Lymphangiogenesic VEGF-C onDry Eye Disease

Dry eye was induced in murine eyes as described in the materials andmethods.

Real time PCR was performed to quantify expression of different VEGFgrowth factors (VEGF-A, VEGF-C, VEGF-D) and their receptors (VEGFR-2,VEGFR-3) in the cornea at days 6, 10 and 14 (FIG. 8) and to determinethe levels of proinflammatory cytokines. IL-1α, IL-1β, IL-6, IL-17 inthe conjunctiva showed significantly decreased expression in anti-VEGF-Ctreated DED mice as compared to those of untreated DED mice (FIG. 9).Draining lymph nodes of anti-VEGF-C treated DED mice showedsignificantly decreased induction of T-cell mediated autoimmune responsecompared untreated DED mice as determined by Real-time PCR analysis forIL-17 (Th17 cells) and IFN-γ (Th1 cells) (FIG. 10).

Enumeration of CD11b⁺/LYVE-1⁺ monocytic cells was done in the DEDcorneas at day 14 as described previously (FIG. 11). Treatment withanti-VEGF-C antibodies significantly decreased infiltration of CD11b⁺cells (30%) in the DED corneas.

To determine whether DED induces growth of lymphatics into the cornea,and whether lymphatic growth is paralleled by growth of blood vessels,corneal whole mounts were double stained for CD31 (pan-endothelialmarker) and LYVE-1 (lymphatic vascular endothelial marker) at days 0, 6,10 and 14 and quantified for lymphangiogenesis as described previously.Lymphatics were seen growing toward the center of DED corneas (FIG. 12).Morphometric analysis showed significant increase in both lymphatic area(P<0.0001) and lymphatic caliber (P<0.02) at day 14 of disease (FIG.13). These lymphatics were not accompanied by any new blood vessels.Lymphangiogenic specific VEGF-D and VEGFR-3 were the earliest toincrease at day 6 followed by increase in VEGF-C, VEGF-A and VEGFR-2.Increased recruitment of CD11b⁺/LYVE-1⁺ monocytic cells to the corneawas also seen with disease.

These results demonstrate that low-grade inflammation associated withdry eye is an inducer of lymphangiogenesis without accompaniedhemangiogenesis.

Clinical Relevance: Demonstration of selective lymphatic growth into dryeye corneas provides an important mechanistic link to adaptive (Tcell-meditated) immunity by delineating how corneal antigen traffickingcan occur to the lymphoid tissues.

Dry eye disease (DED) once thought to be solely due to deficiency oftears, is increasingly being recognized as an immune-mediated disorder)DED affects many millions of people with a wide spectrum of seminalfeatures ranging from mild ocular discomfort to sight-threateningcorneal complications such as persistent epithelial defects and sterilestromal ulceration) In the United States alone, more than 3.2 millionwomen and 1.6 million men above the age of 50 years are affected by thispotentially disabling disease adversely impacting the vision-relatedquality of life.

Clinically significant DED is associated with ocular surfaceinflammation, although the precise immunopathogenesis is not known.There is strong evidence regarding T cell involvement in thepathogenesis of DED in both animal models and humans. Recently, weillustrated T cell activation in the regional lymph nodes of dry eyemice, coincident with acquisition of specific chemokine markers whichhelp in the homing of T cells to the inflamed ocular surface. Further wedemonstrated induction of autoimmunity in the draining lymph nodes ofdry eye mice due to impaired Treg function and generation of pathogenicTh17 cells. These Th17 cells were found to be resistant to Treg mediatedsuppression, leading to unrestrained generation of pathogenic T cellsand sustained ocular surface inflammation. Accordingly, much of the workto date has focused on understanding immunological phenomena occurringin the lymphoid compartment and the effector responses therebygenerated, leaving unanswered the question as to how naive T cells inthe draining lymph nodes get primed to the ocular surface antigen(s)that drive immunity in DED.

The draining lymph nodes are critical sites for induction of immunityand their role in generation of alloimmunity has been well establishedin corneal transplantation. The enhanced survival rate of cornealtransplants in mice with excised cervical lymph nodes implicates theimportance of functional flow of antigen presenting cells (APCs) fromthe ocular surface to the to the draining lymphoid tissue as a necessarycomponent of alloimmunity and graft rejection. However, little is knownabout the pathway that allows trafficking of corneal APCs to thedraining lymph nodes where they prime naive T cells to corneal antigensand generate autoimmune responses in dry eye.

Emphasis is now being given to the importance of pathologicalangiogenesis (hem- and lymphangiogenesis) in various corneal diseasessuch as different forms of keratitis, chemical burns, graft vs hostdisease etc., but to date there is no data regarding cornealangiogenesis in DED. A plausible reason could be that most of the abovementioned conditions except DED are accompanied by in-growth ofclinically visible blood vessels into the cornea. Traditionally it hasbeen thought that lymphatics and blood vessels which serve as afferentand efferent arms of the immune response respectively are alwayscoexistent in pathological states. The present work provides the firstevidence for selective lymphangiogenesis occurring in DED cornea using amurine model. Herein, we attempt to determine the growth of lymphaticvessels into the cornea with the progression of DED, discuss thepathophysiologic implications of corneal lymphangiogenesis in dry eyeand the potential of antilymphangiogenic therapy for ameliorating DED.

DISCUSSION

Lymphangiogenesis in the postnatal period is primarily a response toinflammation and is seen in various pathological states as diverse astumor metastasis, wound healing and transplantation. Lymphatics play animportant role in generating immuno-inflammatory responses by directingthe antigen bearing immunocytes (e.g. dendritic cells) from theperiphery to the draining lymph nodes where T cells are primed andexpanded. The normal human cornea is avascular, thus suppressing theafferent lymphatic and efferent vascular arms of the immune cycleInflammation however negates this “immune” and “angiogenic” privilegedstate of the cornea and gives it the potential to mount an immuneresponse.

Angiogenesis in the cornea is now extensively being studied in variouspathological models such as transplantation. Whereas corneal bloodvessels have long been thought to be an important risk factor for immunerejection in corneal transplantation, it is only recently afterunveiling of new lymphatic specific markers, that the significance oflymphangiogenesis in corneal alloimmunity has been characterized.Despite recognizing the role of inflammatory angiogenesis in the eye,little has hitherto been studied regarding angiogenic mechanisms in DED.Desiccating stress in DED initiates an immune-based inflammatoryresponse that is sustained by the ongoing interplay between the ocularsurface and various pathogenic immune cells, primarily the CD4⁺ T cellsin the conjunctiva and CDI 1b÷ monocytic cells in the cornea.Desiccating stress induces secretion of inflammatory cytokines,especially interleukin (IL)-1, tumor necrosis factor-a, and IL-6 byocular surface tissues, which facilitate the activation and migration ofresident APCs toward the regional draining LN. Our data on frequenciesof mature APC in the LN also suggest increased trafficking of mature APCin

the LN of DED mice (FIG. 7). In the LN, these APCs stimulate naive Tcells, leading to the

expansion of IL-17 secreting Th17 cells and interferon (IFN)-y-secretingTh1 cells. Once these effectors are generated in the LN, they migrate tothe ocular surface and secrete effector cytokines. Recent work hasprovided evidence for the induction of T cell mediated autoimmuneresponses in the regional lymph nodes of DED mice. But what has remainedunanswered is how corneal APCs can traffic to the draining lymphoidcompartment in order to initiate the immune cycle in DED.

Interestingly, to date there has been no published data on thisimportant facet of immunity in DED. The data presented herein clearlydemonstrates the development of lymphatic vessels in the setting of thedry eye state. These lymphatic vessels increase both in caliber and areawhile advancing toward the corneal center with progression of dry eye.Remarkably, these lymphatic vessels are not accompanied by growth ofblood vessels. Various spatio-temporal studies examining relationbetween new blood and lymphatic vessels have led to the belief that apreexisting blood vascular bed is necessary to guide lymphangiogenesis.The current study refutes the general perception of wound healing modelsin skin where growth of lymphatic vessels follows that of blood vesselsby several days. This is also in contrast to other robust models ofcorneal inflammation where there is either parallel outgrowth of bloodand lymphatic vessels or the blood vessels are precedent over thelymphatics. This provides the first evidence of selective ‘natural’ (nonpharmacologically induced) lymphangiogeneis in a disease model that isdissociated from hemangiogenesis.

Lymphangiogenesis is mediated primarily by the interaction of growthfactors VEGF-C and VEGF-D on VEGFR-2 and VEGFR-3. VEGF-A alsocontributes, albeit indirectly, to lymphangiogenesis by recruitingVEGF-C and VEGF-D secreting macrophages. In the present study, dry eyeinduction led to the up-regulation of all the VEGF growth factors andtheir receptors. Though the rise in levels of VEGF-A, VEGF-C and VEGFR-2occurred at later time points (day 14), it is noteworthy, that VEGF-Dand VEGFR-3 (which are both largely specific to lymphangiogenesis)increased as early as day 6 of disease. The functional relevance of theearly rise of VEGF-D is highlighted in a recent study where VEGF-D, viaits action on VEGFR-3, was shown to be a critical modifier of VEGF-Cdriven early sprouting and migration of lymphatic endothelial cells.Macrophages also seem to play a crucial role in lymphangiogenesis. Undernormal physiological conditions, all ocular tissues except the centralcornea are rich in bone marrow derived LYVE-1⁺ macrophages which mayserve as precursor cells for de novo formation of lymphatics. In thepresent study, we noticed significantly increased number ofCD11b⁺/LYVE-1⁺ cells in the peripheral corneas after exposure todesiccating stress, suggesting that either these cells infiltrate intoor multiply from pre-existing CD1 113′7 LYVE-1+ cells in the cornea, andcontribute to lymphangiogenesis. Alternatively, there is a possibilityof upregulation of LYVE-1 in the previous CD11b⁺/LYVE-1⁻ cells.

In summary, presented herein is novel evidence for the selective growthof lymphatic (but not blood) vessels in dry eye disease providing newinsights into the pathophysiology of the disease. The findings suggestthat these newly formed corneal lymphatics may serve as potentialconduits for migration of corneal APCs to lymphoid tissues where theygenerate autoreactive Th17 and Th1 cells in DED. This study not onlyprovides a link between ocular surface inflammation and the generationof T cell mediated immunity in the lymphoid compartment, but also offersan example of how lymphangiogenesis and hemangiogenesis can be‘naturally’ dissociated in a pathological state. The severing of the‘eye-lymphatic axis’ in other immune-mediated conditions, such astransplant rejection, has been shown to hold promise as a strategy ofsuppressing alloimmunity without inhibiting needed innate host defensemechanisms. Similarly, a strategy targeting prolymphangiogenic factorssuch as VEGF-C or VEGF-D may prove effective in ameliorating dry eyedisease.

Example 7 Blockade of Prolymphangiogenic VEGF-C Suppresses Dry EyeDisease

Effect of in vivo blockade of pro-lymphangiogenic VEGF-C on Dry EyeDisease Rationale: Dry eye disease (DED) is an immune-mediated disorderwhose precise pathogenesis remains largely unknown. While it has beenclearly established that in DED generation of pathogenic CD4⁺ T cells(Th1/Th17) primarily occur in the draining lymph nodes, the mechanismsof trafficking of corneal antigen presenting cells (APC) to lymphoidtissues where they activate and expand pathogenic CD4⁺ T cell subsets,were still not well understood prior to the invention described herein.The present invention provides evidence for the selective growth oflymphatic (but not blood) vessels in DED cornea. Data shows asignificant increase in both caliber and extent of lymphatics in DEDcorneas which was also confirmed using real-time PCR by showing a highlysignificant over-expression of lymphangiogenic receptor VEGFR-3 (incontrast to a non-statistically significant increase in hemangiogenicreceptor VEGFR-2 expression). This study not only provides a linkbetween ocular surface inflammation and the generation of T-cellmediated immunity in the lymphoid compartment, but also offers anexample of how lymphangiogenesis and hemangiogenesis can be ‘naturally’dissociated in a pathological state. Data suggests that these corneallymphatics may serve as conduits for migration of corneal APCs tolymphoid tissues where they activate autoreactive T cells in DED.

Immunopathogenesis of DED: The pathogenesis is not fully understood.Ocular surface inflammation sustained by ongoing activation andinfiltration of pathogenic immune cells. Strong evidence of T cellinvolvement. Recent work draining lymphoid tissue primary site foractivation and generation of auto reactive effector T cells in DED(Chauhan et al; Role of cTh17 cells in the immunopathogenesis of dry eyedisease. Mucosal Immunol. 2009; 2(4):375-376.).

Expression levels of VEGF's and VEGFR's in DE corneas using RT PCR hasdemonstrated an increased transcript expression of VEGF-C, VEGF-D, andVEGFR-3. Thus, targeting pro-lymphangiogenic VEGF-C/D has therapeuticimplications in DED.

Corneal lymphatics play an important role in mediating the cornealinflammation in dry eyes. Experiments: To validate this, inhibition ofcorneal neolymphangiogenesis was performed in a well characterized mousemodel of DED described above. To see if inhibition of cornealneolymphangiogenesis could decrease ocular surface inflammation,anti-VEGF-C antibodies were administered i.p. daily from day −1 to day10 to DED mice and assessed clinically using corneal fluoresceinstaining.

Methods (as described previously): Induction of Dry Eye Disease.Experimental Dry Eye Murine Model. Assessment of Corneal Surface:Corneal Fluorescein Staining. Immunohistochemistry: Monocyte/macrophagemarker—CD11b; Pan-endothelial marker—CD31; Lymphatic endothelialmarker—LYVE-1; Blood vessels: CD31^(hi)/LYVE-1; Lymph vessels:CD31^(lo)/LYVE-1^(hi). Morphometry of Lymphangiogenesis: Automated imageanalysis program written using Mat lab. Lymphatic Area (LA)—totalsurface area of the lymphatic vessels when projected into the plane ofthe image. Lymphatic Caliber (LC)—measure of the diameters of thelymphatic vessels.

Anti-VEGF-C antibody and treatment regimen. Experimental design: Threegroups: Normal, DE group treated with IP normal Saline (Untreated) andDE group treated with anti-VEGF-C antibody (VGX-100; a gift fromVegenics, Australia). Daily IP application of anti-VEGF-Cantibody/Normal saline from day −1 to day 13. Dose: 400 pg (20 mg/kg) in100 pl of Normal Saline.

The results are presented in FIG. 14. Results: The data clearly shows asignificant decrease in disease severity in anti-VEGF-C-treated groupcompared to the untreated group. In conclusion, suppression of lymphaticgrowth with VEGF-C blockade led to significant improvement in DEDreflected by decrease in: corneal epitheliopathy; corneal infiltrationof CD11b⁺ cells; expression of pro-lymphangiogenic growth factors andreceptors (VEGF-C, -D, R3) in DE corneas; and mRNA expression levels ofpro-inflammatory cytokines in the conjunctiva.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. While specificembodiments of the subject invention have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe invention will become apparent to those skilled in the art uponreview of this specification. The full scope of the invention should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations. Suchequivalents are intended to be encompassed by the following claims.

1. A method of treating dry eye disease (DED) in a human comprising:administering a composition comprising a tyrosine kinase inhibitor thatinhibits the activity of VEGFR-3 and a pharmaceutically acceptablecarrier to the human subject, in an amount effective to treat dry eyedisease.
 2. The method of claim 1, wherein the composition isadministered to the eye of the human. 3-5. (canceled)
 6. The method ofclaim 1, wherein the DED is an autoimmune DED or a DED associated withSjogren's syndrome.
 7. The method of claim 1, wherein the DED is DED dueto excessively fast tear evaporation (evaporative dry eyes) orinadequate tear production.
 8. The method of claim 1, wherein the dryeye disease is attributable to one or more causes selected from: aging,contact lens usage and medication usage.
 9. The method of claim 1,wherein the dry eye disease is a complication of LASIK refractivesurgery. 10-24. (canceled)
 25. The method of claim 1, further comprisingadministering an anti-inflammatory agent to the subject.
 26. The methodof claim 25, further comprising administering cyclosporine to thesubject.
 27. The method of claim 1, wherein said composition furthercomprises a molecule that inhibits an activity of an inflammatorycytokine selected from the group consisting of IL-1, IL-7, IL23, IL-6and TNF-α.
 28. The method of claim 1, wherein the method furthercomprises administering an antibiotic to the human.
 29. (canceled) 30.The method of claim 28, wherein the antibiotic is selected from thegroup consisting of amikacin, gentamicin, kanamycin, neomycin,netilmicin, streptomycin, tobramycin, teicoplanin, vancomycin,azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, troleandomycin, amoxicillin, ampicillin, azlocillin,carbenicillin, clozacillin, dicloxacillin, flucozacillin, meziocillin,nafcillin, penicillin, piperacillin, ticarcillin, bacitracin, colistin,polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin,lomefloxacin, moxifloxacin, norfloxacin, oflazacin, trovafloxacin,mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole,trimethoprim, cotrimoxazole, demeclocycline, soxycycline, minocycline,oxytetracycline, or tetracycline.
 31. The method of claim 2, wherein theeye comprises a tissue or gland in or around the eye selected from thegroup consisting of ocular tissue, eyelids of the subject, ocularsurface, meibomian gland and or lacrimal gland of the human.
 32. Themethod of claim 1, wherein said composition is administered topically tothe eye of the human subject.
 33. The method of claim 1, wherein thecomposition is formulated for topical administration.
 34. The method ofclaim 1, wherein said composition is in the form of a solid, a paste, anointment, a gel, a liquid, an aerosol, a mist, a polymer, a film, anemulsion, or a suspension.
 35. The method of claim 1, wherein thecomposition further comprises a compound selected from the groupconsisting of physiological acceptable salt, poloxamer analogs withcarbopol, carbopol/hydroxypropyl methyl cellulose (RP MC),carbopol-methyl cellulose, carboxymethylcellulose (CMC), hyaluronicacid, cyclodextrin, and petroleum.